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FLAVONOIDS: BIOSYNTHESIS,
BIOLOGICAL EFFECTS
AND DIETARY SOURCES
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NUTRITION AND DIET RESEARCH PROGRESS SERIES
Flavonoids: Biosynthesis,
Biological Effects and Dietary Sources
Raymond B. Keller
2009. ISBN: 978-1-60741-622-7
NUTRITION AND DIET RESEARCH PROGRESS SERIES
FLAVONOIDS: BIOSYNTHESIS,
BIOLOGICAL EFFECTS
AND DIETARY SOURCES
RAYMOND B. KELLER
EDITOR
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Preface vii
Chapter 1 Bioavailability and Metabolism of Dietary Flavonoids –
Much Known – Much More to Discover 1
David E. Stevenson, Arjan Scheepens and Roger D. Hurst
Chapter 2 Cytoprotective Activity of Flavonoids in Relation
to Their Chemical Structures and Physicochemical Properties 53
Jingli Zhang and Margot A. Skinner
Chapter 3 Oligomeric Nature, Colloidal State, Rheology,
Antioxidant Capacity and Antiviral Activity of Polyflavonoids 97
A.Pizzi
Chapter 4 Grapefruit Flavonoids: Naringin and Naringinin 141
Ricky W. K. Wong and A. Bakr M. Rabie
Chapter 5 Development of Promising Naturally Derived
Molecules to Improve Therapeutic Strategies 181
Dominique Delmas, Frédéric Mazué, Didier Colin,
Patrick Dutartre and Norbert Latruffe
Chapter 6 Effect of a Diet Rich in Cocoa Flavonoids on
Experimental Acute Inflammation 213
M. Castell, A. Franch, S. Ramos-Romero,
E. Ramiro-Puig, F. J. Pérez-Cano and C. Castellote
Chapter 7 Mechanisms at the Root of Flavonoid Action in Cancer:
A Step Toward Solving the Rubik's Cube 231
Maria Marino and Pamela Bulzomi
Chapter 8 Antiophidian Mechanisms of Medicinal Plants 249
Rafael da Silva Melo, Nicole Moreira Farrapo,
Dimas dos Santos Rocha Junior, Magali Glauzer Silva,
José Carlos Cogo, Cháriston André Dal Belo,
Léa Rodrigues-Simioni, Francisco Carlos Groppo
and Yoko Oshima-Franco
vi Contents
There is no doubt that some unconjugated flavonoids do get into the circulation at low
concentrations, but they are quantitatively swamped by the bulk of flavonoid conjugates and
colonic metabolites.
In contrast with the much-studied metabolism of flavonoids, relatively little is known
about the biological activities of their conjugates and metabolites, although what is known
comes from a predominance of in vitro studies and suggests that the metabolites do have
numerous and significant biological activities. Hence very little is known about the real and
mostly indirect benefits and mechanisms of action of dietary flavonoids.
In this review, the current state of knowledge in this area is discussed, with the aim of
stimulating further research (especially by intervention studies) to aid greater understanding.
Chapter 2 - Flavonoids are widely distributed in fruit and vegetables and form part of the
human diet. These compounds are thought to be a contributing factor to the health benefits of
fruit and vegetables in part because of their antioxidant activities. Despite the extensive use of
chemical antioxidant assays to assess the activity of flavonoids and other natural products that
are safe to consume, their ability to predict an in vivo health benefit is debateable. Some are
carried out at non-physiological pH and temperature, most take no account of partitioning
between hydrophilic and lipophilic environments, and none of them takes into account
bioavailability, uptake and metabolism of antioxidant compounds and the biological
component that is targeted for protection. However, biological systems are far more complex
and dietary antioxidants may function via multiple mechanisms. It is critical to consider
moving from using ‘the test tube’ to employing cell-based assays for screening foods,
phytochemicals and other consumed natural products for their potential biological activity.
The question then remains as to which cell models to use. Human immortalized cell lines
derived from many different cell types from a wide range of anatomical sites are available
and are established well-characterized models.
The cytoprotection assay was developed to be a more biologically relevant measurement
than the chemically defined antioxidant activity assay because it uses human cells as a
substrate and therefore accounts for some aspects of uptake, metabolism and location of
flavonoids within cells. Knowledge of structure activity relationships in the cytoprotection
assay may be helpful in assessing potential in vivo cellular protective effects of flavonoids.
This chapter will discuss the cytoprotective properties of flavonoids and focuses on the
relationship between their cytoprotective activity, physicochemical properties such as
lipophilicity (log P) and bond dissociation enthalpies (BDE), and their chemical structures.
The factors influencing the ability of flavonoids to protect human gut cells are discussed, and
these support the contention that the partition coefficients of flavonoids as well as their rate of
reaction with the relevant radicals help define the protective abilities in cellular environments.
By comparing the geometries of several flavonoids, its possible to explain the structural
dependency of the antioxidant action of these flavonoids.
Chapter 3 - The determination by Matrix-Assisted Laser Desorption/Ionization time–of-
flight (MALDI-TOF) mass spectroscopy of the oligomeric nature of the two major industrial
polyflvonoid tannins which exist, namely mimosa and quebracho tannins, and some of their
modified derivatives indicates that: (i) mimosa tannin is predominantly composed of
prorobinetinidins while quebracho is predominantly composed of profisetinidins, that (ii)
mimosa tannin is heavily branched due to the presence of considerable proportions of
"angular" units in its structure while quebracho tannin is almost completely linear. These
structural differences also contribute to the considerable differences in viscoity of water
Preface ix
solutions of the two tannins. (iii) the interflavonoid link is more easily hydrolysable, and does
appear to sometime hydrolyse in quebracho tannin and profisetinidins, partly due to the linear
structure of this tannin, and confirming NMR findings that this tannin is subject to
polymerisation/depolymerisation equilibria. This tannin hydrolysis does not appear to occur
in mimosa tannin in which the interflavonoid link is completely stable to hydrolysis. (iv)
Sulphitation has been shown to influence the detachment of catechol B-rings much more than
pyrogallol-type B-rings. (vi) The distribution of tannin oligomers, and the tannins number
average degree of polymerisation obtained by MALDI-TOF, up to nonamers and decamers,
appear to compare well with the results obtained by other techniques. As regards procyanidin
tannins, it has been possible to determine for mangrove polyflavonoid tannins that: (i)
procyanidins oligomers formed by catechin/epicatechin, epigallocatechin and epicatechin
gallate monomers are present in great proportions. (ii) oligomers, up to nonamers, in which
the repeating unit at 528-529 Da is a catechin gallate dimer that has lost both the gallic acid
residues and an hydroxy group are the predominant species. (iii) oligomers of the two types
covalently linked to each other also occur.
Water solution of non-purified polyflavonoid extracts appear to be incolloidal state, this
being due mainly to the hydrocolloid gums extracted with the tannin as well as to the tannin
itself.
Commercial, industrially produced mimosa, quebracho, pine and pecan polyflavonoid
tannin extracts water solutions of different concentrations behave mainly as viscous liquids at
the concentrations which are generally used for their main industrial applications. Clear
indications of viscoelastic response are also noticeable, among these the cross-over of the
elastic and viscous moduli curves at the lower concentrations of the range investigated, with
some differences being noticeable between each tannin and the others, pine and quebracho
tannin extracts showing the more marked viscoelastic behaviour. Other than pH dependence
(and related structural considerations), the parameters which were found to be of interest as
regards the noticeable viscoelastic behaviour of the tannin extracts were the existence in the
solutions of labile microstructures which can be broken by applied shear. This is supported by
the well known thixotropic behaviour of concentrated, commercial polyflavonoid tannin
extracts water solutions.
Such microstructures appear to be due or (i) to the known colloidal interactions of these
materials, or (ii) to other types of secondary interactions between tannin oligomers and
particularly between tannin and carbohydrate oligomers. The latter is supported by the
dependence of this effect from both the average molecular masses of the tannin and of the
carbohydrate oligomers.
The behaviour of polyflavonoid tannins as regards their antioxydant capacity and radical
scavenging ability has been examined. Radical formation and radical decay reactions of some
polyflavonoid and hydrolysable tannins has been followed, and comparative kinetics
determined, for both light induced radicals and by radical transfer from a less stable chemical
species to the tannin as part of an investigation of the role of tannin as antioxidants.
The five parameters which appear to have a bearing on the very complex pattern of the
rates of tannin radical formation and radical decay were found to be (i) the extent of the
colloidal state of the tannin in solution (ii) the stereochemical structure at the interflavonoid
units linkage (iii) the ease of heterocyclic pyran ring opening, (iv) the relative numbers of A-
and B-rings hydroxy groups and (v) solvation effects when the tannin is in solution. It is the
x Raymond B. Keller
combination of these five factors which appears to determine the behaviour as an antioxidant
of a particular tannin under a set of application conditions.
The chapter ends with some recent results on the antiviral activity of polyflavonoid
tannins for a great variety of viruses.
Chapter 4 - Naringin is the flavonoid compound found in grapefruit that gives grapefruit
its characteristic bitter flavor. Grapefruit processors attempt to select fruits with a low
naringin content, and often blend juices obtained from different grapefruit varieties to obtain
the desired degree of bitterness. Naringin is believed to enhance our perception of taste by
stimulating the taste buds; some people consume a small amount of grapefruit juice before a
meal for this reason.
Naringin and its aglycone naringinin are commonly used health supplements; they exert a
variety of biological actions. This article attempts to review their pharmacokinetics and
pharmacological actions from scientific publications up to November 2008 including effects
on the cardiovascular system, on the skeletal system, on smooth muscle, on the gastric
intestinal system, on the endocrine system, also effects against tumours, protection against
toxins in chemotherapy drugs and the environment, antioxidant effects, drug interactions,
anti-inflammatory effects, and the newly discovered osteogenic and antibacterial actions.
Chapter 5 - Numerous epidemiological studies show that some nutrients may protect
against vascular diseases, cancers and associated inflammatory effects. Consequently, the use
of phytoconstituents, namely those from the human diet, as therapeutic drugs is relevant.
Various studies report the efficiency of phyto-molecules which have cellular targets similar to
those of the new drugs developed by pharmaceutical companies. Indeed, more than 1600
patents are currently reported concerning flavonoids and 3000 patents concerning
polyphenols. Pleiotropic pharmaceutical activities are claimed in fields such as cancer,
inflammation arthritis, eye diseases and many other domains. The increase of activities after
combination with other natural compounds or therapeutic drugs is also patented. In addition,
the aforementioned molecules, from natural origin, generally exhibit low toxicities and often
a multipotency which allow them to be able to simultaneously interfere with several
signalling pathways. However, several in vivo studies revealed that polyphenols / flavonoids
are efficiently absorbed by the organism, but unfortunately have a low level of bioavailability,
glucuronidation and sulphation being limiting factors. Therefore, many laboratories are
developing elements to increase bioavailability and consequently the biological effects of
these natural molecules. For example, the modifications in the lipophilicity of molecules
increase the cellular uptake and consequently involve a best absorption without loss of their
activities. Moreover isomerisation and methylation of hydroxyl groups of polyphenols (e.g.
resveratrol) change the cell molecular targets and are crucial to improve the molecule
efficiency in blocking cell proliferation. In this review, the focus is on the relevance of using
flavonoid and polyphenol combinations or chemical modifications to enhance their biological
effects. Innovative directions to develop a new type of drugs which may especially be used in
combination with other natural components or pharmacological conventional drugs in order
to obtain synergistic effects is discussed.
Chapter 6 - Cocoa has recently become an object of interest due to its high content of
flavonoids, mainly the monomers epicatechin and catechin and various polymers derived
from these monomers called procyanidins. Previous in vitro studies have shown the ability of
cocoa flavonoids to down-regulate inflammatory mediators produced by stimulated
macrophages, but there are no studies that consider the effects of in vivo cocoa intake on
Preface xi
inflammatory response. The present article, reports the in vivo cocoa inhibitory effect on the
acute inflammatory response. Female Wistar rats received Natural Forastero cocoa containing
21.2 mg flavonoids/g for 7 days (2.4 or 4.8 g per rat kg, p.o.). Then, acute inflammation was
induced by means of carrageenin, histamine, serotonin, bradykinin or PGE2 hind-paw
injection. Rats fed 4.8 g/kg/day cocoa showed a significant reduction in the hind-paw edema
induced by carrageenin from the first hour after induction (P<0.05). However, cocoa intake
did not modify the edema induced by histamine, serotonin or PGE2. Only a certain protective
effect was observed at the lowest dose of cocoa in the bradykinin model. Moreover,
peritoneal macrophages from rats that received 4.8 g/kg/day cocoa for 7 days showed a
reduced ability to produce radical oxygen species (ROS), nitric oxide (NO), tumor necrosis
factor α (TNFα) and interleukin 6 (IL-6). This fact could justify, at least partially, the
beneficial effect of cocoa on carrageenin-induced inflammation. In summary, a diet rich in
cocoa flavonoids was able to down-regulate the acute inflammatory response by decreasing
the inflammatory potential of macrophages.
Chapter 7 - The biological activity of flavonoids was first recognized when the
antiestrogenic principle present in red clover that caused infertility in sheep in Western
Australia was discovered. These adverse effects of flavonoids placed these substances in the
class of endocrine-disrupting chemicals. On the other hand, flavonoids are recently claimed to
prevent several cancer types and to reduce incidence of cardiovascular diseases, osteoporosis,
neurodegenerative diseases, as well as chronic and acute inflammation. Despite these
controversial effects, a huge number of plant extracts or mixtures, containing varying
amounts of isolated flavonoids, are commercially available on the market as dietary
supplements and healthy products. The commercial success of these supplements is evident,
even though the activity and mechanisms of flavonoid action are still unclear.
Owing to their chemical structure, the most obvious feature of flavonoids is their ability
to quench free radicals. However, in the last few years many exciting new indication in
elucidating the mechanisms of flavonoid actions have been published. Flavonoids inhibit
several signal transduction-involved kinases and affect protein functions via competitive or
allosteric interactions. Among others, flavonoids interact with and affect the cellular
responses mediated by estrogen receptors (ERα and ERβ). In particular, our recent data
indicate that some flavonoids (i.e., naringenin and quercetin) decouple specific ERα action
mechanisms, important for cell proliferation, driving cells to the apoptosis. Therefore, distinct
complex mechanisms of actions, possibly interacting one another, for nutritional molecules
on cell signalling and response can be hypothesized.
Aim of this review is to provide an updating picture about mechanisms by which
flavonoids play a role in cellular response and in preventing human pathologies such as
cancer. In particular, their direct interaction with nuclear receptors and/or by their ability to
modulate the activity of key enzymes involved in cell signaling and antioxidant responses
will be presented and discussed.
Chapter 8 - Vegetal extracts usually have a large diversity of bioactive compounds
showing several pharmacological activities, including antiophidian properties. In this study,
both coumarin and tannic acid (100 µg/mL) showed no changes in the basal response of
twitches in mouse nerve phrenic diaphragm preparations. In opposite, Crotalus durissus
terrificus (Cdt 15 µg/mL) or Bothrops jararacussu (Bjssu 40 µg/mL) venoms caused
irreversible neuromuscular blockade. Tannic acid (preincubated with the venoms), but not
coumarin, was able to significantly inhibit (p<0.05) the impairment of the muscle strength
xii Raymond B. Keller
induced by Cdt (88 ± 8%) and Bjssu (79 ± 7.5%), respectively. A remarkable precipitation
was observed when the venoms were preincubated with tannic acid, but not with coumarin.
Plathymenia reticulata is a good source of tannins and flavonoids whereas Mikania laevigata
contain high amounts of coumarin. P. reticulata (PrHE, 0.06 mg/mL) and M. laevigata
(MlHE, 1 mg/mL) hydroalcoholic extracts were assayed with or without Bjssu or Cdt
venoms. Both PrHE and MlHE showed protection against Bjssu (79.3 ± 9.5% and 65 ± 8%,
respectively) and Cdt (73.2 ± 6.7% and 95 ± 7%, respectively) neuromuscular blockade. In
order to observe if the protective mechanism could be induced by protein precipitation,
tannins were eliminated from both extracts and the assay was repeated. MlHE protected
against the blockade induced by Bjssu (57.2 ± 6.7%), but not against Cdt. The conclusion is
that plants containing tannins could induce the precipitation of venoms’ proteins and plants
containing coumarin showed activity against Bothrops venoms, but not against Crotalus
venoms. Also concluded is that the use of isolated bioactive compounds could not represent
the better strategy against ophidian venoms, since the purification may exclude some
bioactive components resulting in a loss of antivenom activity. In addition, M. laevigata
showed better antiophidian activity than P. reticulata.
Chapter 9 - There are serious concerns about the increasing global cancer incidence. As
currently used chemotherapeutics agents often show severe toxicity in normal cells, anti-
carcinogenic compounds included in the dietary intakes of natural foods are expected to be
applicable to a novel approach to preventing certain types of cancer without side effects.
Polyphenolic compounds, such as flavonoids, are ubiquitous in plants and are presently
considered to be the most promising in terms of having anti-carcinogenic properties probably
due to their antioxidant effect. To gain further insights into how flavonoids exert anti-
carcinogenic actions on cancer cells at the molecular level, many intensive investigations
have been performed. Currently, the common signaling pathways elicited by flavonoids are
recognized as tumor suppressor p53 and survival factor AKT. These factors are potential
effectors of flavonoid-induced apoptosis via activation of Bax and caspase family genes. The
present chapter emphasizes pivotal molecular mechanisms underlying flavonoid-induced
apoptosis in human cancer cells. In particular, this chapter focuses on representative
flavonoids such as soy isoflavone, green tea catechin, quercetin, and anthocyanin.
Chapter 10 - Flavan-3-ols with the most well known members being catechin and
epicatechin are a group of phenolic compounds widely distributed in nature. The oligomers
and polymers of flavan-3-ols are known as condensed tannins which used to be considered as
anti-nutritional components. In recent years, more and more evidences proved that these
compounds were beneficial to human health as nutrition and lifestyle have fundamentally
changed in modern society. These phenolic compounds showed great potential for the
treatment of lifestyle related diseases, such as type 2 diabetes, obesity, and metabolic
syndrome. They were also reported to have effects on slowing down the aging progress as
well as on prevention of Alzheimer’s disease, cardiovascular disease, and cancer. This
chapter describes the structures, chemical properties, isolation and identification methods,
bioactivity and distribution of flavan-3-ol monomers and condensed tannins in dietary sources
and medicinal plants. Case studies such as the chemical and biological investigations of
tannins in the stems of Cynomorium songaricum (a well known tonic in China) and in other
plants are provided.
Chapter 11 - Accurate taxonomic groupings are important for many applications,
especially for medicinal plants. For example, structural analogues of the antitumour agent,
Preface xiii
paclitaxel, found in common Taxus species have increased the availability of this life-saving
medicine without relying on the slow growing and comparatively uncommon, T. brevifolia
[1]. Flavonoids have a long history of use as chemotaxonomic markers and have assisted in
resolving many taxonomic disputes that have arisen as a result of morphological
classification. In recent times, there has been increased interest in using molecular systematics
and bioinformatics as alternatives to traditional chemotaxonomic techniques, however the
investigation of the types flavonoids present in plants is still a useful technique to rapidly
assess plant taxonomy.
Planchonia careya is a medicinal plant that contains a range of antibacterial compounds.
Species of this genus are morphologically related to Barringtonia and Careya species and
there have been several changes of nomenclature to reflect the uncertainty of these
relationships.
Our recent investigation of some of the comprising flavonoids from Planchonia careya
has revealed similar distinctive compounds to those found in Planchonia grandis that are
notably absent from Barringtonia and Careya taxa. Therefore the comparatively simple
analysis of the flavonoid component of plant extracts can confirm or contest phenetic
groupings to help resolve taxonomic discrepancies.
Chapter 12 - y ubiquitously occur in all parts of plants including the fruit, pollen, roots
and heartwood. Plant extracts rich in flavonoids such as: Ginkgo biloba extract, soy bean
extract and green tea extract are popular dietary supplements. Numerous physiological
activities have been attributed to them and their potential roles in the prevention of hormone-
dependent cancers have been investigated. Over 5000 different flavonoids have been
described to date and they are classified into at least 10 chemical groups. Flavonoid
compounds usually occur in plants as glycosides in which one or more of the phenolic
hydroxyl groups are combined with sugar residues.
Quality control of these plant-extract products is problematic due to the many varying
factors in herbal medicine. Unlike synthetic drugs (in which the concentration and activity of
a single known bio-molecule is monitored), there are many uncertainties in terms of species
variation, geographical source, cultivation, harvest, storage and processing techniques which
may lead to a product of different quality and efficacy. To evaluate the quality of flavonoids
contained within plant extracts it is therefore very important to develop an analytical method
which can monitor the quantity and variety of flavonoids efficiently. Also the study of the
absorption and retention of these compounds within individuals is more problematic as more
than one active compound is ingested from the plant extracts.
The challenge presented by such extracts is therefore to determine the different flavonoid
species present in any given extract and also to determine any modification or fortification of
the extract. Furthermore, Administration, Distribution, Metabolism and Excretion (ADME)
studies require the quantitative analysis of multiple components rather than a single drug
compound. The increase in the number of new flavonoid reports is due to two main factors:
the advances in methods of separation and the rapid development and application of modern
mass spectrometry (MS). Mass spectrometry proved to be the most effective technique in
flavonoids research both in plant extract and in biological samples. This expert commentary ll
reviews the methods available for studying this wide-ranging class of compounds and also
how modern techniques have been applied in order to "mine" the data obtained for flavonoid
specific information from a complex metabolomic analysis.
xiv Raymond B. Keller
Chapter 1
ABSTRACT
There have been many epidemiological studies linking flavonoid intake to health
benefits and many in vitro studies demonstrating various biological effects of flavonoids
that should be reflected by health benefits. It has been widely assumed that these
observations are linked and dietary flavonoids are readily absorbed into the circulation
and influence many regulatory and signalling pathways in tissues. More recently, it has
become apparent that only a small proportion of dietary flavonoid intake is actually
absorbed directly and measured relative absorption varies about 2 orders of magnitude
between different compounds.
It is also apparent that most of the dietary load of flavonoids finds its way to the
colon where the numerous and varied microflora metabolise them into simpler but much
more bioavailable compounds. Further complications to the bioavailability of flavonoids
are added by human Phase II conjugative metabolism, which is thought to convert most
absorbed flavonoids into polar conjugates.
There is no doubt that some unconjugated flavonoids do get into the circulation at
low concentrations, but they are quantitatively swamped by the bulk of flavonoid
conjugates and colonic metabolites.
In contrast with the much-studied metabolism of flavonoids, relatively little is known
about the biological activities of their conjugates and metabolites, although what is
∗
Correspondence: Dr. David E Stevenson. Tel: +64 7 959 4485. Fax: +64 7 959 4431, E-Mail:
dstevenson@hortresearch.co.nz
∗
Correspondence: Dr. Jingli Zhang. Tel: ++64 9 9257100, Fax: ++64 9 9257001, Email jzhang@hort research.co.nz
2 David E. Stevenson, Arjan Scheepens and Roger D. Hurst
known comes from a predominance of in vitro studies and suggests that the metabolites
do have numerous and significant biological activities. Hence we actually know very
little about the real and mostly indirect benefits and mechanisms of action of dietary
flavonoids.
In this review, we discuss the current state of knowledge in this area, with the aim of
stimulating further research (especially by intervention studies) to aid greater
understanding.
INTRODUCTION
Numerous epidemiological trials have associated dietary flavonoid intake with health
benefits, such as reductions in incidence of cardiovascular disease, stroke, diabetes and some
types of cancer [1-12].
In addition, numerous in vitro studies have linked flavonoids to biological effects that
could reasonably underlie the observed health benefits [13]. Although it is very tempting to
assume a simple relationship between the two observations, decades of research into the
absorption, distribution, metabolism and excretion (ADME) of flavonoids strongly
contradicts this simplistic idea.
Research into this field started in the 1950s and was so prolific that in 1991, Scheline was
able to include over 100 studies (mostly in animals) on flavonoid metabolism in his
comprehensive “Handbook of mammalian metabolism of plant compounds” [14]. The main
conclusion from all this work was that, unlike pharmaceutical compounds, which are mainly
found relatively intact, as glucuronide derivatives in plasma or urine, flavonoids were much
more extensively metabolised.
In addition to glucuronides, sulphates and methyl conjugates, the main urinary
metabolites found were phenolic acids of the type also found to be produced by
biotransformation using intestinal bacteria. A few studies using, 14C-labelled flavonoids,
found significant or even extensive degradation into simple compounds such as acetate,
butyrate and carbon dioxide.
It appeared that the major part of the dietary flavonoid intake was not absorbed directly,
but found its way into the colon where the extensive and varied microflora metabolised the
flavonoids into simpler and apparently more bioavailable compounds. Examples of structures
of the major compound classes discussed here are shown in Figure 1 (common flavonoids)
and Figure 2 (common metabolites).
In recent years, much more detail has been added and we now have a relatively good
understanding of flavonoid ADME. It appears that only a tiny proportion (approx 1-2%) of
most dietary flavonoids is actually available to tissues and cells.
Most is metabolised by colonic microflora and most of the remainder that is absorbed
through the gut is conjugated, mainly by glucuronidation, but also by sulphation and
methylation.
The distribution of flavonoids is made more complex by factors like binding of
flavonoids to serum proteins, such as albumin and the activities of cellular efflux pumps
expressed by some cell types.
Bioavailability and Metabolism of Dietary Flavonoids 3
OH O
O OH
Glucose OH O
OH O
OH OH
HO OH
OH OH
O
HO O+ HO O OH
HO
O OH HO OH OH
Glucose
OH OH
O
OH
Cyanidin-3-glucoside Epicatechin
HO
HO OH OH
OH
O
OH OH
HO OH
HO HO
OH
OH O
OH Procyanidin C1
Resveratrol Phloretin
There have been many excellent reviews discussing various aspects of these findings [15-
30], but it is only relatively recently that serious attention has been given to determination of
the biological activities of flavonoid metabolites and comparison with the parent compounds.
In contrast to the many hundreds of published studies on the biological effects of the
quantitatively very minor species absorbed in vivo, i.e., flavonoid aglycones, published
studies on metabolites only number around 50 at the time of writing.
We still, therefore, have little idea of the actual, as opposed to potential, relative
contributions to health of flavonoid aglycones, flavonoid conjugates and colonic metabolites.
We have attempted to bring together all available information to date, to stimulate further
research into the biology of flavonoid metabolites.
Our overall understanding of flavonoid ADME is summarised in Figure 3 and the chapter
is organised to follow flavonoids through the various stages of digestion, absorption,
distribution and metabolism and cover what is known about each stage.
The last section describes what we currently understand about the biological activities of
the metabolites.
4 David E. Stevenson, Arjan Scheepens and Roger D. Hurst
O OH
OH OH
O OH OH
OH O
HO O O
O
OH
O H O HO S
N CH O
2 C
OH OH
OH O
OH O
OH O
HO HO
OH OH OH
HO
O O O
3,4-dihydroxybenzoic acid 4-hydroxybenzoic acid Benzoic acid
(Protocatechuic; PCA)
HO HO
HO O
HO
O OH O OH
OH
3,4-dihydroxyphenylacetic acid 4-hydroxyphenylacetic acid Equol (a major metabolite of genistein)
HO HO HO OH
OH OH
HO
O O OH
Figure 2. Structures of some flavonoid conjugates and colon flora-derived metabolites. Many other
isomers of the phenolic acids have been detected, e.g., 2-, 3-, and 4-hydroxybenzoic acid and also O-
methylated metabolites.
Me
+Bil
thyl
Glu
UGT
/sul
Colon + Micro flora
cu
ron
pha
id
te
es
Flavonoid Absorption?
Flavonoid aglycones?
aglycones
Phenolic acids Liver
[Phase II
Kidney enzymes]
Phenolic
acids Faeces Urine
Human saliva was found to have significant, but highly variable hydrolytic activity [32],
and hence hydrolysis of flavonoid glucosides may start in the oral cavity. It was not clear
whether detached epithelial cells or bacteria, or both are the source of glucosidases. The
resulting aglycones may be absorbed from the stomach, most likely by passive transport,
whereas glycosides appear only to be absorbed by the intestine [33].
The Stomach
It is unclear whether procyanidins are metabolised in the stomach. In one study under
simulated stomach acid conditions, procyanidins (epicatechin polymers, trimers to hexamers)
have been found to be partially broken down, primarily to monomers and dimers [34]. This
6 David E. Stevenson, Arjan Scheepens and Roger D. Hurst
would be expected to enhance both bioavailability via intestinal absorption and colonic
metabolism. In contrast, however, another study found that procyanidins were stable in the
stomach [35], suggesting that they should be predominantly available to colonic flora.
Quercetin was absorbed in the rat stomach whereas its glycosides were not [33]. This
suggests absorption by passive diffusion and the probable relative absence of suitable
transporters or glycosidases. This result is the opposite of what is generally found in the
intestine (see below). Anthocyanins at the very high concentration of 750 μM were absorbed
directly from rat stomach to the extent of ~20% of the administered dose over 30 minutes
[36]. The stomach is therefore clearly capable of anthocyanin absorption, but the
physiological relevance of the high concentration used is questionable. Investigation of
raspberry fruit extracts in an in vitro digestion model [37] suggested that anthocyanins and
other flavonoids bind reversibly to other food components (protein, polysaccharides etc) and
that this may increase their stability. This binding to polymers appears to modify the
absorption and excretion of anthocyanins, but not their metabolism in rats [38], or pigs [39].
conjugated and pumped back out [42]. This suggests that efflux pumps can limit
bioavailability by returning flavonoid conjugates to the apical side/lumen, but cannot
completely prevent some material from reaching the basolateral side/circulation.
The overall picture that emerges from the numerous bioavailability/metabolism studies is
that there are essentially three groups of flavonoids with distinctively different behaviour,
anthocyanins, procyanidins/catechins and “everything else”. The aglycone forms of common
flavonoids, such as quercetin, naringenin, or daidzein (and probably less common but
structurally similar flavonoids) have been tested in many studies and are consistently the best-
absorbed. These flavonoids are generally both more hydrophobic and more stable than
anthocyanins or catechins and this may account for their higher observed plasma
concentrations. Single doses routinely give plasma Cmax values in the low micromolar range
in humans (Table 1). Long-term dosing of pigs with quercetin gave similar results to single
doses (~1 μM) [43], but in rats, plasma concentrations were over 20 μM, suggesting that pigs
are probably a better model of human physiology [44]. Flavonoid aglycones, however, are
rare in plant foods, glycosides usually being the only form found, with the exception of the
relatively polar catechins, which are rarely glycosylated but, nevertheless, apparently much
less bioavailable than hydrophobic compounds like quercetin. Where flavonoid aglycones are
found in foods (specifically quercetin in onion skin), they appear to be more bioavailable than
even glucoside derivatives [45].
The nature of glycosylation has an important influence on absorption. A number of
studies have demonstrated the abundance of β-glucosidase, but the absence of other
glycosidase activities in the GI tract and its tissues. Two β-glucosidases capable of
hydrolysing flavonoid glycosides were found in human small intestinal mucosa. Lactase-
phloridzin hydrolase (LPH) was localized in the apical membrane of gut epithelial cells while
β-glucosidase was found in the cytosol [46]. This suggests that LPH can facilitate absorption
by deglycosylating flavonoids and promoting passive diffusion, whereas the cytosolic enzyme
can only act after absorption of the glucoside. Ovine lactase phloridzin hydrolase, a β-
glucosidase found on the brush border of the mammalian small intestine, was readily able to
hydrolyse glucosides of quercetin, genistein and daidzein [47]. Intestinal and liver β-
glucosidases readily cleaved flavonoid glucosides, but not other glycosides [48]. There is
some evidence from the studies in Table 2, that quercetin glucosides and maybe other
flavonoid glucosides, can be transported intact by glucose transporters, but the main
mechanism of glucoside absorption appears to be deglycosylation by membrane-bound β-
glucosidase, followed by relatively rapid diffusion of the aglycone across the cell membrane
[49-51]. Other glycosides may diffuse slowly across cell membranes [52-55], but the major
proportion appears to pass into the colon, where microflora expressing various glycosidases
can liberate the aglycones for absorption. It appears, for example, that a relatively high
proportion of quercetin from the glucoside is absorbed from the intestine, as its plasma
concentration peaks in 1-2 hours and it is predominantly cleared in 5-6 hours. In contrast,
Rutin is absorbed much more slowly [56], to a much lower Cmax, but most is deglycosylated
in and absorbed from the colon as quercetin, giving a low but more sustained plasma level of
quercetin conjugates that can last more than 24 hours.
Intestinal epithelial cells have high glucuronyl transferase activity and appear to
glucuronidate ~95% of flavonoids before transfer into the bloodstream, or efflux back into the
lumen [57-60].
Table 1. Summary of recent published studies involving determination of plasma/tissue bioavailability (Cmax) from oral ingestion of
flavonoids or flavonoid-rich foods and /or identification of major detected metabolites
Grapefruit extract Dog Plasma Cmax 0.3-0.4 μM, primarily naringin, with small proportions of naringenin and [98]
its glucuronide.
Hesperetin, naringenin Human Plasma Max concentrations 3 and 7 μM respectively, mostly as conjugated forms. [99]
(aglycones) 135 mg each Phenolic acid metabolites also detected.
Isoflavones (glucosides of Human Intact isoflavones are absorbed in 2 “peaks”; apparently the first from direct intestinal [100]
genistein and daidzein) absorption and the second from the colon after microbial metabolism.
Kaempferol (a flavonol, like Human Even a low dose (9 mg) resulted in a peak plasma concentration of 0.1 μM and urinary [101]
quercetin) excretion of 2% of dose. Suggests that bioavailability higher than quercetin.
Luteolin and related Rat Nobiletin much better absorbed and widely distributed in tissues. Major metabolites [64]
permethylated flavone, Nobiletin were 3 mono-demethylated and 2 di-demethylated forms.
Naringenin and its glucoside Rat Orally, ~10% of both compounds absorbed and mainly recovered as glucuronide. [102]
Intravenously, glucoside recovered largely unchanged. Glucoside readily cleaved and
glucuronidated during intestinal absorption.
Naringenin, hesperetin Human Peak plasma concentrations reached after 5 hours of ~0.1-0.2 μM, 95% detected as [103]
glycosides from Blood orange conjugates.
juice
Normal diet Human Thirteen polyphenols and metabolites were found suitable as biomarkers of polyphenol [104]
intake; cinnamic acids: chlorogenic acid, caffeic acid, m-coumaric acid, gallic acid, 4-
O-methylgallic acid; flavonoids: quercetin, isorhamnetin, kaempferol, hesperetin,
naringenin, phloretin; lignans: enterolactone, enterodiol. Suggests that cinnamic acids,
hydrophobic flavonoids and lignans are the most bioavailable phenolics.
Pelargonidin aglycone by oral Rat 18% of dose absorbed after 2 hours and detected in liver, kidney, brain, lung but not [73]
gavage heart, spleen. Main metabolites glucuronide and 4-hydroxybenzoic acid.
Phloretin, quercetin and their Rat intestine in situ Perfusion of glucosides or aglycones generated same mixture of conjugates in blood. [105]
glucosides Glucosylation increased quercetin absorption, but reduced phloretin absorption.
Polymethoxylated flavone Rat Plasma metabolites all glucuronides of parent compound or demethylated forms. [106]
mixture isolated from Chinese
herb
Procyanidin dimers Isolated rat intestine 95% absorbed as unconjugated epicatechin, i.e., the dimers were cleaved into [107]
monomers by the intestinal cells.
Procyanidin-rich chocolate Human Plasma C max of epicatechin range from 0.13-0.35 μM. [108]
Flavonoid/Source Species Bioavailability in plasma/tissues; Metabolites detected Reference
Procyanidins and catechins from Rat Conjugates of catechins only, no evidence of absorption or degradation of [109]
oral grape seed extract procyanidins.
Procyanidins and catechins from Rat No evidence of procyanidin absorption, only gallic acid and catechin monomers [110]
oral grape seed extract detected.
Puerarin (daidzein-8-C- Rat Un-metabolised puerarin plasma conc 6 μM after 4 hours, detected in brain extracts. [111]
glucoside) Metabolites detected: hydroxylated and reduced forms including equol.
Quercetin Rat intestine Quercetin rapidly absorbed, but ~ half glucuronidated and excreted back in to lumen. [59]
Quercetin Rat Quercetin conjugates (no aglycone) appeared in lymphatic fluid, with the maximum [112]
concentration at 30 minutes.
Quercetin aglycone from Shallot Human Cmax 1μM from flesh and 4μM from skin; suggests that food matrix increases aglycone [45]
skin, quercetin glucosides solubility/absorption compared with pure compound.
(shallot flesh/1.4 mg/kg total
quercetin
Quercetin and its glucoside Isolated rat intestine Glucoside absorbed faster than aglycone, via hexose transporter, but deglycosylated [49]
during or after absorption. Only aglycone and glucuronides found in mucosal tissue.
Quercetin glycosides Rat intestine Deglycosylation and absorption of quercetin aglycone was much higher from [50]
glucosides than other glycosides.
Quercetin glycosides from Human Quercetin-3-glucuronide, 3'-methyl-quercetin-3-glucuronide and quercetin-3'-sulfate, [113, 114]
onions no source glycosides detected.
Quercetin glycosides from Human Five isomeric quercetin glucuronides. [115]
onions
Quercetin in diet Pig Single dose of 25 mg/kg (50 mg/kg/day/4 weeks) resulted in μM concentrations of: [43]
plasma 1.6 (0.8), liver 0.03 (0.15), muscle 0.3 nM (both), brain (0.07 nM).
Quercetin mono- and di- Human Sub-micromolar maximum concentrations of intestinal conjugates (glucuronide, [116]
glucoside from fried onions sulphate) after~40 min and peak of liver metabolite (sulpho-glucuronide) after 2.5
hours. Considerable differences between plasma and urinary conjugate profiles. Total
urinary excretion 4.7%.
Quercetin, catechin, red wine Rat Only quercetin could be detected in plasma, as conjugates. None of the supplements [117]
polyphenols, orally reduced lipid peroxidation markers.
Quercetin, long-term dietary Rat, Pig Rats, 0.1% quercetin in diet/11 weeks resulted in ~20 μM total quercetin (inc. [44]
supplementation conjugates) in plasma and 0-4 μM in organs; pigs, 500mg/kg in diet/3 days resulted in
1.25 μM in plasma, 6 μM in liver and 2.5 μM in kidney.
Quercetin, oral/6 weeks Rat 11 different conjugates after absorption, major one a methyl, sulpho, glucuronide. [70]
Table 1. (Continued)
Various efflux pumps have been implicated in the return of glucuronidated flavonoids to
the lumen [61, 62]. The material returned to the lumen is thought to pass into the colon and to
14 David E. Stevenson, Arjan Scheepens and Roger D. Hurst
subsequently become available for microbial metabolism. The positional isomer distribution
of absorbed glucuronides may be influenced not only by the positional selectivity of intestinal
epithelial cell glucuronyl transferase iso-forms, but also by selective efflux back into the
lumen. The MRP2 efflux pump shows selectivity between quercetin glucuronide isomers [63]
and this may contribute to the observed isomer distribution in the circulation. Some
flavonoids, primarily from citrus fruits (e.g., Nobiletin, Sinensetin, Tangeretin) are
permethylated, i.e., they have O-methyl groups in place of phenolic hydroxyl groups. Other
flavonoids can also be easily methylated chemically and permethylated flavonoids have
considerably different ADME properties from other flavonoids [64-68]. Methylation
increases hydrophobicity and therefore intestinal absorption. Blocking of all potential
conjugation sites (i.e., phenols) by methylation inhibits Phase II conjugative metabolism,
requiring Phase I, cytochrome P450-mediated demethylation before conjugation is possible
[66]. These findings suggest an approach to counteract the issue of low flavonoid
bioavailability for health studies (assuming that it is desirable), and also highlight the
effectiveness of mammalian cell metabolism at limiting the absorption of flavonoids.
The array of flavonoid conjugates from gut cell metabolism appears to be further
expanded by the liver, which appears to accumulate higher concentrations of flavonoids than
any other organ [44]. Liver β-glucuronidase can apparently remove glucuronidation added by
the intestinal cells and sulpho-transferase and catechol O-methyl transferase (COMT) can
substitute (or add) sulphation and/or methylation. For example, quercetin is primarily
glucuronidated by intestinal cells, before passage into the bloodstream. When cultured
hepatocytes were treated with purified quercetin glucuronides to investigate potential further
metabolism in the liver, three major processes were observed. Firstly, methylation of the
quercetin moiety of the glucuronides by COMT was noted before removal of glucuronyl
residues by β-glucuronidase, followed by sulphation [69]. The potential for a complex
conjugate mixture resulting from conjugation by both intestine and liver was highlighted by a
rat study with quercetin [70]. Eleven conjugates were detected, the most abundant having all
3 conjugate groups, i.e., methyl, glucuronide and sulphate, in the same molecule.
Anthocyanins are very unstable compared with other flavonoids, particularly at neutral or
alkaline pH. The anthocyanin content of a wide variety of processed blackcurrant products
was only 0.05-10% of that in fresh fruit. Although they should be relatively stable in the
acidic environment of the stomach, degradation in the neutral small intestine may be very
rapid [71]. Bioavailability studies on anthocyanins consistently find barely detectable
concentrations in plasma (<0.05 μM) with only a tiny proportion of the dose (often <0.1%)
excreted in urine (Tables 1, 4). This low urinary excretion compares with up to ~20% for
some other flavonoids (see below). Elevated levels of phenolic acid metabolites can account
for only a low percentage of intake (Tables 1, 4). It is becoming clear from the numerous
bioavailability studies on anthocyanins that 60-90% of the dietary load disappears from the
GI tract within 4 hours of a meal [72]. It is not clear, however, what happens to them.
Possibilities are that they are degraded/structurally rearranged into as-yet unidentified
derivatives, or covalently bound to proteins or other polymers (i.e., resistant to
extraction/detection), either in the intestinal lumen, plasma, or inside cells. Anthocyanin
detection is commonly achieved using very sensitive techniques such as liquid
chromatography-mass spectroscopy (LC-MS). However, recoveries from anthocyanin
extraction procedures performed from plasma are often very low (less than 20%) and hence it
Bioavailability and Metabolism of Dietary Flavonoids 15
is possible that the apparently low bioavailability of anthocyanins is partly due to a lack of
robust techniques to enable complete extraction and detection.
Anthocyanins are unusual in that, unlike other flavonoids, the predominant forms
detected in plasma and urine are intact, un-metabolised glycosides, rather than aglycones or
conjugates. They may be very unstable under physiological conditions after deglycosylation,
because, apart from the strawberry anthocyanin, pelargonidin [73], there is very little
evidence of significant deglycosylation or conjugation of anthocyanins. Although it may
appear that anthocyanins probably do not have significant biological activities because of
their apparently low bioavailability, there is some evidence that absorption from the stomach
[74] and the jejunum [75], may be both rapid and relatively high. Particularly rapid
degradation after absorption, however, appears to severely limit the duration of significant
anthocyanin plasma concentrations, in normal circumstances. Only traces survive long
enough to be detected in the urine. Localised biological effects of anthocyanins immediately
after absorption, or direct effects on the gastrointestinal tract or its microflora are possible,
however.
Colonic microflora display an extensive capability to metabolise flavonoids that reach the
colon either directly, or after intestinal epithelial cell metabolism and apical efflux of
glucuronides, biliary excretion etc. Numerous studies (Table 3) have demonstrated the ability
of isolated cultures, faecal slurries, etc, to remove glycosylation that is resistant to
endogenous mammalian β-glucosidases and cleave flavonoids into simpler compounds. As
with the published bioavailability studies, quercetin is the most studied and best understood.
It appears that quercetin glucosides, for example, are relatively well absorbed in the small
intestine (see above discussion), whereas the glucosidase-resistant rutin reaches the colon
mostly intact where it can be deglycosylated by microflora making it potentially available for
both absorption by colonic epithelial cells and degradation into simpler compounds. One
report proposed a mechanism to explain the observed degradation of quercetin by pig caecum
microflora, predominantly into phloroglucinol and 3,4-dihydroxyphenylacetic acid [143].
These degradation products appear to accumulate in the colon to concentrations 1-2 orders of
magnitude higher than the flavonoid aglycones [144]. There is evidence from urinary studies
[145-148], that the phenolic acid degradation products, particularly benzoic acid, a likely final
product of these degradation pathways, are well absorbed and increased urinary excretion
correlates with consumption of flavonoid-rich foods. Hippuric acid (benzoylglycine) is
regularly detected at high concentrations in urine, following consumption of flavonoid-rich
foods and is known to be the major human conjugate of benzoic acid [146]. Studies in vitro
using Caco-2 and other gut epithelial cells (Table 2) have demonstrated that these low
molecular weight acids are efficiently transported, possibly by a mono-carboxylic acid
transporter present in the colonic epithelia and so rapid absorption through the gut wall is not
unexpected. One recent study found various small aromatic compounds in urine resulting
from quercetin consumption and in addition reported for the first time, mercapturic acid
conjugates of phenolic acids in the urine, apparently derived from colonic breakdown of
glutathione conjugates of quercetin that had been detected in the plasma [149].
16 David E. Stevenson, Arjan Scheepens and Roger D. Hurst
It was proposed that the glutathione conjugates arose from a reaction between a quinone
form of quercetin (resulting from oxidation) and glutathione. This is thought to occur both
spontaneously and catalysed by glutathione-S-transferase.
Although it is clear that flavonoids can be degraded into phenolic acids, there are
alternative dietary sources which make the picture more complicated. Blueberry fruits in
18 David E. Stevenson, Arjan Scheepens and Roger D. Hurst
particular (and presumably plant foods in general), contain large amounts of phenolic acid
compounds bound to insoluble polymeric plant cell wall material, i.e., “fibre”. Simple esters
between phenolic acids and other compounds, including, for example, caffeic-quinic acid
esters (chlorogenic acid) are common in apples and coffee. p-Coumaric-tartaric acid esters
(coutaric acid) are found at reasonable levels in grapes. Unbound acids are a relatively small
proportion of the total phenolic acids present in the edible parts of plants. The bound or
conjugated acids are predominantly hydroxy-benzoic and hydroxy-cinnamic acids and 12
were detected in hydrolysed blueberry fruit fibre [150]. When this insoluble fraction of
blueberry was incubated with human faecal slurry, over 20 phenolic acids were detected,
some clearly liberated unchanged from the fruit fibre, others, (phenylacetic acids in
particular), apparently transformed by the faecal bacteria. An alternative source of phenolic
acids may have been transformation of residual flavonoids. A widely occurring
hydroxycinnamic acid, caffeic acid, can be metabolised by human faecal flora into similar
compounds to those derived from flavonoids, i.e., 4-hydroxyphenylpropionic acid and
benzoic acid [151]. Gut microbe-derived cinnamoyl esterases have been shown to be
primarily responsible for the liberation of free phenolic acids from phenolic acid esters in the
colon [152], whilst endogenous mammalian esterase activity can be found throughout the
gastro-intestinal tract [153]. Since there are apparently three potential source materials for the
phenolic acids generated by the colon flora, flavonoids, fibre and cinnamic acids, care must
be taken linking particular phenolic acid metabolites and flavonoids, using animal or human
studies, unless pure flavonoids are administered.
The occurrence of small phenolic acids in plants is also a complicating factor. It has been
claimed that protocatechuic acid (PCA) is the major metabolite of cyanidin glycosides in
humans, based on a trial involving the consumption of blood orange juice (BOJ). PCA was
the main plasma and urinary metabolite detected and a relatively high (compared with other
anthocyanin studies) proportion of anthocyanins (1.2%) were detected in urine [154]. It was
claimed that the PCA came primarily from intestinal microbial metabolism of cyanidin. This
assumption was based on the finding of PCA as a major metabolite of purified anthocyanins
in rats [73, 92]. The former finding is not conclusive, however, because the Tmax (time after
dosing at which Cmax is attained) of PCA was relatively short, at 2 hours (suggesting direct
absorption). BOJ contains a small amount of PCA and its flavanone content (which may also
be metabolised to PCA) is similar to its anthocyanin content [155]. BOJ consumption clearly
results in significant amounts of absorbed PCA, but it does not necessarily derive from
cyanidin.
Beer, unsurprisingly for a fermented product, is a good direct dietary source of small
phenolic acids. Phenolic acids from beer (some of which are the same compounds as colonic
metabolite acids) were readily absorbed in human subjects and the degree of conjugation
varied considerably [156]. 4-Hydroxyphenylacetic acid reached much higher plasma
concentrations (~1 μM) than cinnamic acids, such as ferulic or caffeic acids and was
predominantly non-conjugated. This suggests that simple phenolic acids, both as produced by
colonic microflora and in the diet, would be readily absorbed and may be conjugated to a
much lesser extent than flavonoids. Similarly, consumed free p-coumaric acid is rapidly and
extensively absorbed through the stomach wall and upper intestine using both passive
diffusion and the mono-carboxylic transporter, reaching plasma levels of 165 μM, albeit with
a very short plasma half-life of 10 minutes [157]. In vitro studies using these acids would
Bioavailability and Metabolism of Dietary Flavonoids 19
have more physiological relevance than those on unconjugated flavonoids because of their
higher bioavailability in relatively unaltered states.
It also appears that most of the flavonoids studied so far are not absorbed directly by the
small intestine, based on total urinary excretion of conjugates of the intact flavonoid. The
highest reported proportion of a dose of flavonoid excreted in urine was 20%, for phloridzin-
derived phloretin [158].
Another study reported 10% from phloridzin or phloretin itself [159]. This implies that at
least 80-90% would have passed on to the colon and been available for microfloral
breakdown (or direct absorption contributing to percentage excreted in urine). Commonly
reported percentages of urinary excretion of intact flavonoids are in single figures and those
for anthocyanins rarely exceed 0.1% (Tables 1, 4).
There is some evidence that anthocyanins may be rapidly and relatively well absorbed
from large doses, but their (presumed) instability under physiological conditions results in
very little surviving to be excreted in the urine. It seems unlikely that the absorbed proportion
could often exceed that for phloretin, so the major proportion of dietary anthocyanins would
be potentially able to reach the colon. As discussed above, however, losses to as-yet unknown
destinations or forms, appear to be extensive although a study in rats shows that at least part
of the dietary intake of intact anthocyanins can reach the colon and undergo metabolism by
faecal flora [160].
High-anthocyanin berryfruit extracts (2.5-5% of diet) were fed for 14 weeks. Recoveries
of different individual anthocyanins from faeces were very variable (6-25%). Interestingly, it
was noted that anthocyanin degradation in faecal samples stored at -18ºC was rapid unless
these were pasteurised before storage, suggesting that faecal bacteria were still active during
storage.
An exception to the extensive degradation of flavonoids to phenolic acids are the soy
isoflavones, which are metabolised to modified, but intact flavonoids such as equol or
angolensin [161]. The isoflavones, (as discussed below), are also the best example of
alternative flavonoid metabolism end products produced by individuals with different
populations of gut microflora, where some individual’s flora can produce equol, whereas
others cannot [162].
Limited evidence suggests that the consumption of flavonoids in the diet can modify the
composition of the colonic flora. This can in turn, modify the metabolism of the flavonoids by
the microflora. Soy isoflavone metabolism was compared in children, who had been, or were
being fed either soy- or cows-milk based infant formula. Those currently or recently
consuming infant formula showed differences in metabolism depending on the type of
formula consumed, but there was no difference in older children [163]. This suggests that
dietary flavonoids can influence the composition of the colonic microflora that metabolise
them, but the effect is not long-lasting.
Tea polyphenolics and their colonic metabolites were recently tested for their effects on
the growth of human colonic microfloral cultures [164]. The phenolics and metabolites
generally inhibited bacterial growth, and pathogenic strains were much more severely
affected than commensal strains. This also suggests that dietary flavonoids can beneficially
influence the composition of the colonic microflora.
Table 4. Summary of published studies on urinary metabolites of flavonoids
cerebral cortex and cerebellum, whereas none were detected in plasma. The total amount of
anthocyanins detected was in the order of 0.7 to 0.9 pmol/g brain tissue and included the un-
metabolised compounds: arabinose, galactose and glucose glycosides of cyanidin,
delphinidin, malvidin and peonidin. This experimental method, where the phytochemical-
containing diet is removed from the animals for 24 hours prior to euthanasia, thus allowing
phytochemical clearance from blood, is a convenient way to prevent blood-borne
phytochemical contamination of organs. Similar low levels of anthocyanins (0.25nmol/g brain
tissue) were found in the brains of rats after 14 days of a blackberry fruit supplemented diet
[77]. In this study the anthocyanins consisted primarily of un-metabolised anthocyanins,
except for a methylated peonidin 3-glucoside. In another study where rats were administered
red grape anthocyanins directly into the stomach, the authors found brain levels of intact
anthocyanins of up to 192 ng/g (~0.6 μM) after only 10 minutes, unfortunately the tissues
were not perfused and the unusually high levels of anthocyanins measured were therefore
likely of both brain and blood origin [78].
The BBB has an extensive expression of MDR efflux transporter pumps which remove
many xenobiotics, phytochemicals and drugs from the BBB endothelial cell layer and thereby
prevent access to the brain proper. The most well characterised and possibly most relevant to
the export of phytochemicals from the brain is P-glycoprotein (P-gp), an ATP-driven efflux
pump which has a preference for lipophilic compounds (reviewed in detail in [225]). In vitro
BBB models can be used to elucidate which of the efflux pumps might be responsible for the
export of specific compounds, for example, Youdim et al [227] showed that both quercetin
and naringenin have some level of central nervous system access which is limited by the
activity of BBB efflux pumps. Pre-treatment with a specific P-gp inhibitor demonstrated
some specificity, inhibiting the efflux of naringenin but not quercetin. When their system was
pre-treated with a P-gp inhibitor which also blocks the action of the breast cancer resistance
protein (BCRP) efflux pump, quercetin efflux was also severely limited. These results
indicate that the naringenin is primarily exported by P-gp whereas quercetin is preferentially
exported by the BCRP efflux pump [227].
It is not clear whether the primary mode by which flavonoids cross the BBB is diffusion
or carrier-mediated transport, but whatever the process, they are apparently very susceptible
to export via efflux pumps with some specificity for individual compounds. This efflux
system is, however, not completely effective, as indicated by in vivo studies showing the
retention of flavonoids within brain tissue at the picomolar to low nanomolar range. It is,
therefore, important that in vitro studies evaluating the action of phytochemicals on brain cell
cultures make use of physiologically relevant doses within this low concentration range.
Table 5. (Continued)
In addition to the numerous in vitro studies listed in Table 5, there have been a number of
studies, often in vivo, combining investigation of metabolism and determination of biological
activities of the metabolites.
28 David E. Stevenson, Arjan Scheepens and Roger D. Hurst
Quercetin/Flavonols
Spencer et al investigated the uptake, metabolism and protection from oxidative stress of
quercetin and its major metabolites (3'-O-methyl quercetin, 4'-O-methyl quercetin and
quercetin 7-O-beta-D-glucuronide) in dermal fibroblasts [255]. Uptake and oxidative stress
protection was highest with quercetin itself and lower with methyl derivatives. The
glucuronide was not taken up by the cells and conferred no protection. In that study, quercetin
appeared to be metabolised by the cells to a glutathione conjugate and a quinine derivative.
Quercetin conjugates (glucuronide, sulphate, O-methyl) reduced COX-2 gene expression in
ex vivo human lymphocytes, but a single feeding of human subjects with onions (containing
163.9 mg quercetin 3, 4'-diglucoside, 140.6 mg quercetin 4'-glucoside, and 2.4 mg quercetin
aglycone) had no effect. Plasma quercetin metabolites attained a Cmax of 4 μM [256].
Quercetin has been implicated in the anti-depressant effects of St John’s Wort (SJW).
When tested on rats using a forced swimming test, SJW extract, rutin and the quercetin
metabolite isorhamnetin (3’-methyl quercetin) all exhibited anti-depressant activity after
administration for 9 days; Isorhamnetin being most effective [257]. After eight days
administration of SJW extract, concentrations in plasma and the central nervous system were
9.6 and 1.3 μM for quercetin and conjugates and 7.4 and 2 μM for methylated quercetin and
conjugates.
High acute doses of quercetin glucopyranoside (Isoquercitrin; 100 mg/kg) achieved
quercetin Cmax in the plasma and central nervous system of 16.5 and 2.9 μM, respectively.
Unfortunately, in this study the brains were not perfused prior to analysis and the brain levels
of these compounds are likely overstated due to contamination from blood circulating in the
brain at the time of death. The corresponding Cmax values for methylated quercetin and
conjugates were 10.7 and 2.7 μM. Human subjects fed a soup high in quercetin exhibited
higher plasma quercetin conjugate concentrations and reduced collagen-stimulated platelet
aggregation than controls on low quercetin soup [258].
Antibody staining of quercetin-3-glucuronide showed that it preferentially accumulated
in macrophage-derived foam cells, abundant in human aortic atherosclerotic lesions, but not
normal aorta, where foam cells are rare.
In addition, the glucuronide was taken up by murine macrophages and de-conjugated to
quercetin aglycone. The aglycone suppressed expression of genes involved in foam cell
formation, suggesting that quercetin conjugates may inhibit atherosclerosis [259].
Conjugation, therefore, appears to have the potential to considerably modify the in vivo
biological activity of absorbed flavonoids.
The picture that emerges from the many studies on quercetin is that the aglycone and its
conjugates share many activities, although often with considerably different efficacy. In
addition, some conjugates lose activities or exhibit activities that the aglycone does not. It is
clearly not valid to assume any relationship between in vitro activities of flavonoid aglycones
and in vivo effects from that compound in the diet. In addition, positive results in vitro, even
from verified conjugates, do not necessarily translate into in vivo effects following an acute
dose.
Bioavailability and Metabolism of Dietary Flavonoids 29
Isoflavones
Some individuals produce equol, as a major metabolite of daidzein, whereas others do not
produce detectable equol [162], presumably because of differences in colonic microflora
composition and isoflavone metabolism. Equol has well characterised cardiovascular benefits
[260]. It also inhibits neoplastic cell transformation in vitro, a potential mechanism for the
anti-cancer effect attributed to daidzein [261]. With regard to cancer therapy genistein and
daidzein glucuronides were oestrogenic, but ~10-fold weaker than their aglycones and weakly
activated natural killer (NK) cells against cancer cells [262]. Microarray analysis of the
effects of soy isoflavones on gene expression uncovered a relationship between the ability to
produce equol and a greater expression in oestrogen-responsive genes [263]. Equol appears to
affect gene expression more than daidzein. Dietary equol attenuated weight gain in
ovariectomised rats, but did not prevent bone-loss and had an undesirable uterotrophic effect
[264, 265]. In contrast, a similar study found that equol did prevent bone loss, but the other
major daidzein metabolite, O-desmethylangolensin, did not [266]. In a study on menopausal
women, 135 mg/day for one week of soy isoflavones only improved menopausal symptoms
in individuals who were equol producers. Equol appears to be primarily responsible for the
health benefits of dietary soy isoflavones. High-dose oral equol (400 mg/kg) in
ovariectomised rats produced a mammotropic (stimulatory) effect, suggesting that equol is
weakly oestrogenic [267].
Catechins
A human intervention trial suggested that the increase in flow-mediated arterial dilation
resulting from tea consumption is inversely related to an individual’s ability to methylate tea
flavonoids [6], i.e., methylation decreases biological activity.
Other Flavonoids
Anthocyanins and their suspected colonic phenolic acid metabolites were tested for their
ability to inhibit platelet activation in vitro [268], an ability thought to be beneficial in the
prevention of coronary vascular disease (CVD). Significant inhibition was observed with 1
μM anthocyanins or 10 μM of most of the phenolic acids tested. Notable exceptions, showing
no activity, were hippuric acid and homovanillic acid. A mixture of all the tested compounds,
(probably more representative of the in vivo situation), was the most effective, even at 1 μM
total concentration. Although the phenolic acids required a higher concentration, they are
likely to be present individually at much higher concentrations than anthocyanins in the body.
The “simulated metabolite mixture” of both individually active and inactive compounds
exhibited a strong synergy, although which of the components were synergistic was not
determined.
Some flavonoid conjugates demonstrate anti-inflammatory activity. Myricetin
glucuronide had an anti-inflammatory effect in a carrageenan-induced rat model of
inflammation and showed inhibitory activity for 5-lipoxygenase and cyclo-oxygenase (COX)-
30 David E. Stevenson, Arjan Scheepens and Roger D. Hurst
1 and COX-2 and [269]. Oral administration to rats of 100 mg/Kg of astilbin (a flavanone
rhamnoside) resulted in low micromolar concentrations in plasma of astilbin and a methylated
metabolite, plus a glucuronide in bile only [270]. The methylated metabolite and astilbin had
similar anti-inflammatory effects when injected intra-peritoneally into mice, i.e., ~halving
picryl chloride-induced ear swelling and nearly normalising elevated levels of tumour
necrosis factor α (TNF) and Interferon-γ (IFN- ).
Apple juice phenolic extracts, fermented anaerobically by human faecal flora, had 30-
50% of the Trolox equivalent antioxidant capacity (TEAC) of the unfermented extracts, but in
Caco-2 cell-based antioxidant assays, the fermented extract was significantly better at
inhibiting ROS formation induced by t-BH [271]. The main identified constituent of the
fermented extract were 3,4-dihydroxy- and 4-hydroxy-phenylpropionic acid, phloroglucinol
and 3,4-dihydroxyphenylacetic acid. These data indicate that inhibition of ROS formation by
phenolics is not likely the result of their chemical antioxidant activity but rather, induction of
endogenous cytoprotective mechanisms.
The plant-derived glucuronide baicalin (bicalein-7-glucuronide) is both the major natural
form and the major human conjugate of the flavonoid baicalein [81]. The glucuronide was
able to induce apoptosis in cultured prostate cancer cells [272] and Jurkat leukemic T
lymphocytes [273]. In the latter case, it appeared to act by caspase activation via the
mitochondrial pathway but cytotoxicity for normal peripheral blood mononuclear cells was
much lower. The intestinal absorption of this compound is much lower than its aglycone [81]
but these results demonstrate the biological activity of a flavonoid glucuronide.
CONCLUSION
Although there is considerable quantitative variation between different studies, even of
the same compound, as to the proportions absorbed, excreted, degraded, or metabolised by
different means, some generalisations can be made. It is clear that net intestinal absorption of
a few individual flavonoids may be as high as 10-20% of intake, under experimental
conditions, but under normal circumstances, figures of 1-2% appear more likely and some
compounds may be lower still. The more hydrophobic flavonoids, when consumed as
glucosides, appear to be the best absorbed, usually via enzymic deglycosylation, followed by
diffusion of the aglycone across intestinal cell membranes. More polar compounds, such as
catechins, are relatively poorly absorbed and glycosides other than glucosides are resistant to
deglycosylation and are slowly absorbed, by diffusion or possibly sugar transporters.
Anthocyanins appear to be hardly absorbed at all, based on urinary excretion measurements,
but newer evidence suggests that their absorption may be both relatively extensive and rapid,
but counterbalanced by very rapid degradation and a consequent very short plasma half-life.
The plasma typically appears to contain only traces of most flavonoid aglycones or their
original glycosidic forms; a very high, but somewhat variable, percentage is in the form of the
polar conjugates, glucuronides and sulphates. The flavonoids in plasma appear to be
predominantly bound to serum albumin and maybe other proteins and this, combined with
cellular MDR efflux pumps, limits organ bioavailability, particularly to the brain. The only
apparent exception is the liver, which may actively take up flavonoid glucuronides, to
relatively high concentrations and appears to modify their conjugation. The intestine appears
Bioavailability and Metabolism of Dietary Flavonoids 31
step towards simplifying the whole area of flavonoid biological activity determination. This
approach may be very useful to assist with interpretation of complex ADME data.
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In: Flavonoids: Biosynthesis, Biological Effects… ISBN: 978-1-60741-622-7
Editor: Raymond B. Keller © 2009 Nova Science Publishers, Inc.
Chapter 2
ABSTRACT
Flavonoids are widely distributed in fruit and vegetables and form part of the human
diet. These compounds are thought to be a contributing factor to the health benefits of
fruit and vegetables in part because of their antioxidant activities. Despite the extensive
use of chemical antioxidant assays to assess the activity of flavonoids and other natural
products that are safe to consume, their ability to predict an in vivo health benefit is
debateable. Some are carried out at non-physiological pH and temperature, most take no
account of partitioning between hydrophilic and lipophilic environments, and none of
them takes into account bioavailability, uptake and metabolism of antioxidant compounds
and the biological component that is targeted for protection. However, biological systems
are far more complex and dietary antioxidants may function via multiple mechanisms. It
is critical to consider moving from using ‘the test tube’ to employing cell-based assays
for screening foods, phytochemicals and other consumed natural products for their
potential biological activity. The question then remains as to which cell models to use.
Human immortalized cell lines derived from many different cell types from a wide range
of anatomical sites are available and are established well-characterized models.
The cytoprotection assay was developed to be a more biologically relevant
measurement than the chemically defined antioxidant activity assay because it uses
human cells as a substrate and therefore accounts for some aspects of uptake, metabolism
and location of flavonoids within cells. Knowledge of structure activity relationships in
the cytoprotection assay may be helpful in assessing potential in vivo cellular protective
effects of flavonoids. This chapter will discuss the cytoprotective properties of flavonoids
and focuses on the relationship between their cytoprotective activity, physicochemical
properties such as lipophilicity (log P) and bond dissociation enthalpies (BDE), and their
chemical structures. The factors influencing the ability of flavonoids to protect human gut
cells are discussed, and these support the contention that the partition coefficients of
54 Jingli Zhang and Margot A. Skinner
flavonoids as well as their rate of reaction with the relevant radicals help define the
protective abilities in cellular environments. By comparing the geometries of several
flavonoids, we were able to explain the structural dependency of the antioxidant action of
these flavonoids.
INTRODUCTION
The flavonoids are among the most numerous and widespread natural products found in
plants and have many diverse applications and properties. Over the years, a wide range of
beneficial properties related to human health have been reported. These include effects related
to cancer (Colic and Pavelic, 2000; Eastwood, 1999; Middleton et al., 2000), cardiovascular
diseases (Riemersma et al., 2001), including coronary heart disease (Eastwood, 1999;
Giugliano, 2000; Middleton et al., 2000) and atherosclerosis (Wedworth and Lynch, 1995);
anti-inflammatory effects (Manthey, 2000; Middleton et al., 2000), and other diseases in
which an increase in oxidative stress have been implicated (Diplock et al., 1998; Harborne
and Williams, 2000; Packer et al., 1999). A number of studies have shown that consumption
of fruits and vegetables can reduce the risk of cardiovascular diseases and cancer, potentially
through the biological actions of the phenolic components such as flavonoids.
The precise mechanisms by which flavonoids may protect different cell populations from
oxidative insults are currently unclear. However, potential mechanisms that involve their
classical antioxidant properties, interactions with mitochondria, modulation of intracellular
signalling cascades, and stimulation of adaptive responses have been proposed. The effects of
a flavonoid in a cellular environment may well extend beyond conventional antioxidant
actions. In the cellular environment, the coexistence of other factors such as the
bioavailability of the compound, the effectiveness of the compound within the cell, and the
effectiveness of the compound in the body must also be considered. Therefore, using a cell-
based assay format, these compounds react with cells and provide information regarding the
cellular response, taking into account some aspects of uptake, metabolism, location of
antioxidant compounds within cells and intracellular effects on signalling pathways and
enzyme activity. These effects are likely to be the result of differential modulation of cellular
activities such as signalling pathways, enzyme activity, transport and bioavailability, rather
than simply a result of free radical scavenging. Furthermore, as cells from different
anatomical sites respond differently to both stressors and treatments, it is important to use the
appropriate cell types to test a particular cellular response, rather than a chemically-defined
system for the antioxidant activity. Here, we employed and established cell-based assays
using gut-derived cultured human cell lines. The rationale to use cultured human cell lines
over primary cells is that human primary cells are not readily available, but human
immortalized cell lines derived from many different cell types from a wide range of
anatomical sites are available and are established well-characterized models.
The multiple biological activities of flavonoids as well as their structural diversity make
this class of compounds a rich source for modelling lead compounds with targeted biological
properties. Different classes of flavonoids are not equally physiologically active, presumably
because they are structurally different. Despite the enormous interest in flavonoids and other
polyphenolic compounds as potential protective agents against the development of human
diseases, the real contribution of such compounds to health maintenance and the mechanisms
Cytoprotective Activity of Flavonoids in Relation… 55
through which they act are still unclear. Structure activity relationships (SARs) represent an
attempt to correlate physicochemical or structural descriptors of a set of structurally related
compounds with their biological activities or physical properties. Molecular descriptors
usually include parameters accounting for electronic properties, hydrophobicity, topology,
and steric effects. Activities include chemical and biological measurements. Once developed,
SARs provide predictive models of biological activity and allow the identification of those
molecular parameters responsible for the biological and physicochemical properties. These
may shed light on the mechanism of action.
SARs of flavonoids have been previously reported for scavenging of peroxynitrite,
hydroxyl radical and superoxide, and protection against lipid peroxidation (Chen et al., 2002;
Choi et al., 2002; Cos et al., 1998), inhibition of LDL oxidation (van Acker et al., 1996; Vaya
et al., 2003), and the influence of flavonoid structure on biological systems has also been
investigated, e.g., induction of DNA degradation; growth and proliferation of certain
malignant cells; acute toxicity in isolated rat hepatocytes, and inhibition of gastric H+, K+-
ATPase (Agullo et al., 1997; Moridani et al., 2002; Murakami et al., 1999; Sugihara et al.,
2003).
Figure 1. Diagram of biosynthetic formation of flavonoid backbone from phenylalanine. The basic
flavonoid structure consists of the fused A and C-rings, with the phenyl B-ring attached through its 1′-
position to the 2-position of the C-ring (numbered from the pyran oxygen).
Flavonoids differ in the arrangements of hydroxyl, methoxy, and glycosidic side groups,
and in the configuration of the C-ring that joins the A- and B-rings. These give rise to a
multitude of different compounds (Middleton et al., 2000). In plants, the majority of the
flavonoids are found as glycosides with different sugar groups linked to one or more of the
hydroxyl groups. They are mainly found in the outer parts of the plants, such as leaves,
flowers and fruits, whereas the content in stalks and roots is usually very limited. The
flavonoids located in the upper surface of the leaf or in the epidermal cells have a role to play
in the physiological survival of plants. They contribute to the disease resistance of the plant,
either as constitutive antifungal agents or as induced phytoalexins (Harborne et al., 2000).
Multiple combinations of hydroxyl groups, sugars, oxygen atoms, and methyl groups attached
to the basic ring structural skeleton create the various classes of flavonoids. According to the
configuration of the C-ring, flavonoid can be classified as flavonol, flavonone, flavone,
flavanol, anthocyanidin, chalcone, and isoflavone (Herrmann, 1976; Herrmann, 1989) as
illustrated in Figure 2. It should be noted that chalcones contain an opened C-ring (Dziezak,
1986) and the numbering system for chalcones is reversed. Flavonoids comprise a large group
of secondary plant metabolites. Presently more than 7000 individual compounds are known,
which are based on very few core structures (Fossen and Andersen, 2006; Stack, 1997).
Within each class, individual flavonoids may vary in the number and distribution of hydroxyl
groups as well as in their degree of alkylation or glycosylation.
Flavones and flavonols occur as aglycones in foods. These compounds possess a double
bond between C2 and C3. Flavones are a class of flavonoids based on the backbone of 2-
phenylchromen-4-one, such as chrysin, apigenin, and luteolin. Flavones are lacking the 3-OH
group on the backbone. Flavones are commonly found in fruit skins, celery, and parsley.
Cytoprotective Activity of Flavonoids in Relation… 57
Flavonols are a class of flavonoids that use the 3-hydroxyflavone backbone (3-hydroxy-
2-phenylchromen-4-one (Figure 2). Flavonols are different from flavones in that they possess
a hydroxyl group in the 3-position and can be regarded as 3-hydroxyflavones. The formation
of flavonol and flavone glycosides depends on the action of light; therefore, they are found
mainly in leaves and fruit skins with only trace amounts in parts of plants below the soil
surface (Herrmann, 1976). In general, flavonols occur in the diet as glycosides (Hollman and
Arts, 2000). Flavonols, such as galangin, kaempferol, quercetin, morin, rutin, myricetin, and
isoquercetin, are found in plant-based foods, with onions, apples, berries, kale, and broccoli
having the highest concentrations. Flavonols are present mainly as mono-, di- and
triglycosides. The monoglycosides occur mainly as 3-O-glycosides. In the case of
diglycosides, the two sugar moieties may be linked to the same or two different carbons.
Flavonones and flavononols are characterized by the presence of a saturated C2–C3 bond
and an oxygen atom (carbonyl group) in the 4-position. Thus, flavonones may be referred to
as dihydroflavones. Flavanones, such as hesperidin and naringin, have a more restricted
distribution than other flavonoid compounds and are specific to citrus fruits. Naringin is the
predominant flavanone in grapefruit (Citrus paradisi) (up to 10% of the dry weight) and is
responsible for the bitterness of grapefruit juices. The flavonones in plants are often
glycosylated in the 7-position with disaccharides rutinose and neohesperoside. Both of these
disaccharides are made of rhamnose and glucose and differ only in their linkage type: 1→6
58 Jingli Zhang and Margot A. Skinner
for rutinose and 1→2 for neohesperoside. It is worth noting that flavononol glycosides are
good fungistatic and fungitoxic substances (Kefford and Chandler, 1970). Flavononols differ
from flavanones by having a hydroxyl group in the 3-position and are often referred to as 3-
hydroxyflavonones or dihydroflavonols, such as taxifolin. Flavonones have one centre of
asymmetry in the 2-position, while flavononols possess a second centre of asymmetry in the
3-position.
Among flavonoids, anthocyanins and flavanols are known collectively as flavans because
of lack of the carbonyl group in the 4-position. Flavanols are a class of flavonoids that use the
2-phenyl-3,4-dihydro-2H-chromen-3-ol skeleton. These compounds include catechin,
epicatechin and its derivates. Flavanols are building blocks for proanthocyanidins.
Proanthocyanidins consist of monomeric units of flavans linked through C-C and ether
linkages. Fifteen subclasses of proanthocyanidins have been identified (Porter, 1993),
however, only three appear to be prominent in human foods of plant origin, procyanidins
(epicatechin or catechin polymers), prodelephinidins (epigallocatechin or gallocatechin
polymers) and propelargonidins (epiafselechin or afselechin polymers) or their mixtures (Gu
et al., 2003). These proanthocyanidins are soluble up to a molecular weight of approximately
7000, corresponding to ca. 20 flavan units. The name proanthocyanidins, previously called
leucoanthocyanidins, implies that these are colorless precursors of anthocyanidins. On heating
in acidic solutions, the C-C bond made during formation is cleaved and terminal flavan units
are released from the oligomers as carbocations, which are then oxidized to colored
anthocyanidins by atmospheric oxygen. Anthocyanidins are naturally colored compounds
occurring in the form of glycosides (anthocyanins), the largest group of water-soluble
pigments in the plant kingdom. They are responsible for most of the red, blue, and purple
colors of fruits, vegetables, flowers, and other plant tissues (Harborne et al., 2000).
Anthocyanidins are characterized by having the basic flavylium cation structure and different
substituents on ring B. The electron deficiency of their structure makes anthocyanidins highly
reactive, and their stability is both pH and temperature dependent. Their glycosides are
usually much more stable than the aglycons (Delgado-Vargas et al., 2000). The anthocyanins
are all based chemically on the structure of cyanidin, and all are derived from this base
structure by the addition or subtraction of hydroxyl groups, by the degree of methylation of
these hydroxyl groups, and by the nature and number of sugars and their position on the
aglycon (Harborne, 1962; Harborne and Williams, 2001). In aqueous media, most of the
natural anthocyanins behave as pH indicators, being red at low pH, bluish at intermediate pH,
and colorless at high pH. The nature of the chemical structures that these anthocyanins can
adopt upon changes in pH has been described (Briviba et al., 1993; Brouillard, 1983).
Chalcones are flavonoids lacking a heterocyclic C-ring. The chalcone structure contains
an aromatic ketone that forms the central core for a variety of important biological
compounds. The most common chalcones found in apples and other fruit of the Rosaceae
family are phloretin and phloretin-7-glycoside (phloridzin).
Cytoprotective Activity of Flavonoids in Relation… 59
BDE is the measure of the energy change on bond making or bond breaking and is
defined as the amount of energy required to break a given bond to produce two radical
fragments when the molecule is in the gas phase at 25°C (298.15 °K) (McMurry, 1992).
60 Jingli Zhang and Margot A. Skinner
A:B ⎯⎯⎯
→ A• + B•
BDE
[1]
Bond dissociation energies have long been considered to provide the best quantitative
measure of the stabilities of the radicals formed (Bordwell and Zhang, 1993). Since the rate
constants of this reaction depend largely on the strength of the ArO-H bond (ArOH represents
flavonoid molecule), the BDE of flavonoids is defined by the following equation:
ArOH ⎯⎯⎯
→ ArO• + H•
BDE
[2]
where Hfr is the enthalpy for radicals generated after H abstraction, Hfh is the enthalpy for the
hydrogen atom, -0.49792 hartrees, and Hfp is the enthalpy of the parent molecule (Zhang et
al., 2003a).
The properties of the ArO-H bond appear to be essential to understanding the chemical
and biochemical behaviour of flavonoids. The ArO-H bond must be broken to generate the
truly active species, i.e. the phenoxy radical, in order to exhibit its antioxidant activity. There
are a number of studies, using a diversity of modern experimental and computational tools, on
the determinations of the BDEs of phenolic derivatives (Bordwell et al., 1993; Lucarini and
Pedulli, 1994; van Acker et al., 1996). Their aim was to understand how the strength of the
phenolic bond is affected by nature, position, and number of substituents. BDE can be
experimentally determined in the gas phase using approaches such as radical kinetics, gas-
phase acidity cycles and photoionization mass spectrometry (Berkowitz et al., 1994) and in
solutions using techniques such as photoacoustic calorimetry (PAC) (Mulder et al., 1988),
electrochemical (EC) (Wayner and Parker, 1993), and other measurements (Mahoney and
DaRooge, 1975). It should be noted that neither the EC technique nor the PAC method for
measuring BDEs is a stand-alone method. Both techniques are dependent upon at least one
gas-phase measurement (Wayner et al., 1995). However, all the above-mentioned methods
are limited especially for the larger organic compounds since most of them are not stable in
the gas phase. Moreover, these measurements require very sophisticated instruments. For
these reasons, the number of experimentally known BDEs for flavonoids is very small
(Denisov and Khydyakov, 1987).
Besides these experimental studies, a number of theoretical investigations of varying
degrees of sophistication have also been reported in order to understand the structural factors
determining the stability of the O-H phenolic bond. Both experimental and theoretical results
indicate that the change of the O-H bond strength due to a given substituent is approximately
constant in the variously substituted phenols and that, for each substituent in the ortho, meta,
and para positions, an additive contribution may be derived that can be used to estimate the
bond BDE of polysubstituted phenols for which experimental data are lacking (Wright et al.,
1997; Wright et al., 2001).
As a fundamental chemical parameter (Borges dos Santos and Simoes, 1998), there have
been several types of theoretical methods to estimate O-H BDE (Chipman et al., 1994). The
first is through the additive rule (Wright et al., 2001). Although this is convenient to estimate
Cytoprotective Activity of Flavonoids in Relation… 61
the O-H BDEs for monophenols (Brigati et al., 2002; Lucarini et al., 1996), it has not been
demonstrated as generally effective for catechols (Zhang et al., 2003a). The second is through
semi-empirical quantum chemical calculations by means of intermediate neglect of
differential overlap (INDO) (Pople et al., 1968), modified neglect of diatomic overlap
(MNDO) (Dewar and Thiel, 1977), the Austin Model 1 (AM1) (Dewar et al., 1985), and the
parameterization method 3 (PM3) (Stewart, 1989). The third is through density functional
theory (DFT) (Qin and Wheeler, 1995; Ziegler, 1991) or ab initio molecular dynamics
(Bakalbassis et al., 2001; Car, 2002) calculations.
The parameterization method 3 (PM3) uses nearly the same equations as the AM1
method along with an improved set of parameters. PM3 predicts energies and bond lengths
more accurately than AM1 (Yong, 2001). Several computer programs, such as Gaussian,
Hyperchem, and MOPAC have been developed based on these theories. All these programs
can be employed to perform the calculation of BDEs. In this chapter, in order to investigate
the cytoprotective mechanism, the BDEs of selected flavonoids were calculated using the
PM3 method using the MOPAC2002 program through Chem3D Ultra 2008
(http://www.camsoft.com/). In this chapter, the difference (ΔHf) between the heat of
formation of a parent molecule (Hfp) and that of its phenoxyl radical (Hfr) is used to represent
the BDE. As stated above, the heat of formation of the H atom (Hfh) is treated as a constant in
order to simplify the calculations (Zhang, 1998; Zhang et al., 1999) and can be ignored. Thus,
BDE can be expressed:
Therefore, ΔHf was used in this chapter to approximate BDE and these two terms are
interchangeable.
Lipophilicity (Log P)
Moser, 1983). The partition coefficient is therefore dimensionless, being the quotient of two
concentrations, and it is customary to express them in logarithmic form to base ten, i.e., as log
P because P values commonly range over many orders of magnitude (Fujita et al., 1964). The
logarithm of the partition coefficient, log P, has been successfully used as a hydrophobic
parameter (Leo, 1991).
Pioneering work by Leo and Hansch (Leo et al., 1971) has led to the use of log P in
quantitative structure-activity relation methods (QSAR), as a general description of cell
permeability. In the field of drug development, log P has become a standard property
determined for potential drug molecules (Lipinski et al., 1997). The lipophilicity of the
flavonoids is an important parameter in chemical toxicology as it can indicate metabolic fate,
biological transport properties and intrinsic biological activity (Hansch et al., 2000).
Lipophilicity is of central importance for biological potency as it plays a role in the
interaction of flavonoids with many of the targets in a biological system. Log P probably can
be considered the most informative and successful physicochemical property in biochemistry
and medicinal chemistry (Leo, 1991). Since log P is an additive, constitutive molecular
property, it is possible to estimate the log P value of a molecule from the sum of its
component molecular fragment values (Masuda et al., 1997). Many programs developed to do
this are based on substructure approaches such as ClogP (Leo, 1991; Leo, 1993), KOWWIN
(Meylan and Howard, 1995), AB/LogP (Japertas et al., 2002), ACD/LogP (Buchwald and
Bodor, 1998; Osterberg and Norinder, 2001), and KLOGP (Klopman and Zhu, 2001). The
substructure methods usually require a long calculation time because a large number of
structural parameters need to be taken into account (Mannhold and Petrauskas, 2003). An
alternative approach for the computation of log P is based on additive atomic contributions.
The Ghose-Crippen approach is the most widely used atom-based method (Ghose and
Crippen, 1987). The parameters used in the calculation of log P can be obtained by first
classifying atoms into different types according to their topological environments, which
contribute differently to the global log P value. Several computer programs are developed
based on atomic contribution techniques such as XLOG P (Wang et al., 1997) and SMILOGP
(Convard et al., 1994). Since the whole is more than the sum of its parts, any method of
calculating log P of a molecule from its parts has limitations. Thus, other methods have been
proposed based on calculated molecular properties. Fewer programs are based on a whole-
molecule approach compared with a substructure approach. The most widely available one is
SciLogP Ultra (Bodor et al., 1989). From a theoretical perspective, it is difficult to judge the
validity of any particular method since it depends on the methodology used in data analysis
and algorithm derivation.
are extensively bound to albumin. Flavonoids are able to penetrate tissues, particularly those
in which they are metabolized, but their ability to accumulate within specific target tissues
needs to be further investigated. Flavonoids and their derivatives are eliminated chiefly in
urine and/or bile, and are secreted via the biliary route into the duodenum, where they are
subjected to the action of bacterial enzymes, especially ß-glucuronidase, in the distal segments
of the intestine, after which they may be reabsorbed. This enterohepatic recycling may lead to
a longer presence of polyphenols within the body.
Anthocyanins can be absorbed intact as glycosides. The mechanism of absorption is not
clear. However, it has been found that anthocyanins can serve as ligands for bilitranslocase,
an organic anion membrane carrier found in the epithelial cells of gastric mucosa (Passamonti
et al., 2002). This finding suggested that bilitranslocase could play a role in the bioavailability
of anthocyanins. Considering the physiological implications, the interaction of anthocyanins
with bilitranslocase suggests that, at the gastric level, it could promote the transport of these
compounds from the lumen into the epithelial layers of the gastric mucosa, thus favoring their
transfer to the portal blood, and then, at the hepatic level, from the portal blood into the liver
cell. At this level, anthocyanins could also be transported by other organic anion carriers.
It has been reported that procyanidin dimmer (B2), epicatechin, and catechin were
detected in the plasma of human subjects as early as 30 min (16, 2.61, 0.13 μM, respectively)
after acute cococa consumption and reached maximal concentrations by 2 h (41, 5.92, and
0.16 μM, respectively) (Holt et al. 2002).
CYTOPROTECTION ASSAY
Oxidative stress refers to the cytopathological consequences of an imbalance between the
production of free radicals and the ability of the cell to neutralize them. Reactive oxygen
species (ROS) have been suggested to be a major cause of neurodegenerative disorders, such
as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease (Simonian and Coyle,
1996). Hydrogen peroxide (H2O2) can traverse membranes and exerts cytotoxic effects on
cells in the proximity of those responsible for its production (Halliwell, 1992). Although
H2O2 is not a free radical and has a limited reactivity, it is thought to be the major precursor
of the highly reactive hydroxyl radical (HO•). Recent studies have shown a close association
between H2O2 and neurodegenerative disease, and it has been suggested that H2O2 levels are
increased during pathological conditions such as ischemia (Behl et al., 1994; Hyslop et al.,
1995).
ROS such as H2O2 and HO• readily damage biological molecules that can eventually lead
to apoptotic or necrotic cell death (Gardner et al., 1997). Exposure of cells to oxidative stress
induces a range of cellular events that can result in apoptosis or necrosis (Davies, 1999).
Apoptotic cells can be evaluated based on the measurement of the loss of plasma membrane
asymmetry (van Engeland et al., 1998). Under normal physiological conditions, a cell
maintains a strictly asymmetric distribution of phospholipids in the two leaflets of the cellular
membranes with phosphatidylserine (PS) facing the cytosolic side (Devaux, 1991). However,
during early apoptosis this membrane asymmetry is rapidly lost without concomitant loss of
membrane integrity (van Engeland et al., 1998). Cell surface exposure of PS, which precedes
the loss of membrane integrity, can be detected by fluorescein isothiocyanate (FITC)-labelled
Cytoprotective Activity of Flavonoids in Relation… 65
annexin V, a reagent that has high affinity for PS residues in the presence of millimolar
concentrations of calcium (Ca2+) (Andree et al., 1990). By simultaneous probing of
membrane integrity by means of exclusion of the nuclear dye propidium iodide (PI), apoptotic
cells can be discriminated from necrotic cells (Darzynkiewicz et al., 1997). The importance of
apoptosis in the regulation of cellular homeostasis has mandated the development of accurate
assays capable of measuring this process. Apoptosis assays based on flow cytometry have
proven particularly useful; they are rapid, quantitative, and provide an individual cell-based
mode of analysis (rather than a bulk population).
In this chapter, the relationship between physicochemical properties, chemical structures
and cytoprotective capacity of twenty-four different flavonoids was established using human
colon adenocarcinoma (HT-29) cells.
Cell viability was assessed using the WST-1 assay. The WST-1 assay is based on the
cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases to form dark red
formazan, which absorbs at 450 nm. Cultured human cells were seeded in 96-well plates (in
0.1 ml medium) at a density of 5 ×105 cells/ml with various concentrations (0.25-20 μM) of
testing compounds. Tested compounds were either dissolved in DMSO or deionised water
depending on their solubility (the amount of DMSO in cell culture was limited to 0.1%).
Equivalent amounts of the DMSO vehicle had no effect compared with results in control
cells. After 24 h of incubation in a humidified 5% CO2, 95% air atmosphere at 37°C, 10 μl
WST-1 tetrazolium salt was added to each well and the cells were incubated for 2 h to allow
the reaction between the mitochondrial dehydrogenase released from viable cells and the
tetrazolium salt of the WST-1 reagent.
The absorbance was measured at 450 nm with a reference at 690 nm using a microplate
reader (Synergy HT, BioTEK Instruments, Winooski, VT). The level of absorbance directly
correlates to viable cell numbers. Each assay was performed in triplicate and the cell viability
was expressed as a percentage of the absorbance of cells exposed to test samples compared
with that of controls (cells only).
66 Jingli Zhang and Margot A. Skinner
HT-29 cells were grown in McCoy's 5A medium (modified) supplemented with 10%
fetal bovine serum (FBS) in the presence of 100 U/ml penicillin and 0.1 g/l streptomycin at
37°C in humidified air with 5% CO2. Cultured HT-29 cells were plated in 24-well plates at a
density of 5 × 105 cells/ml. A range of non-toxic concentrations (0-20 μM) of testing
compounds were added together with 150 μM H2O2. Cells were incubated for 24 h at 37°C in
humidified air with 5% CO2.
Calculation of Results
Figure 3. Cytograms of control (prior to incubation) (A), incubated control (B) and 150 μM H2O2
treated (C) human colon adenocarcinoma (HT-29) cells.
The viable cells are located in the lower left corner (negative in both annexin V-FITC and
propidium iodide) (A3). Early apoptotic cells are in the lower right corner (annexin V-FITC
Cytoprotective Activity of Flavonoids in Relation… 67
positive only) (A4). Late apoptotic cells that show progressive cellular membrane and nuclear
damage are in the upper right corner (both annexin V-FITC and PI positive) (A2). Necrotic
cells are located in the upper left corner (PI positive only) (A1). The total percentage of
damaged cells (both apoptotic and necrotic) was considered as (A1+A2+A4). The cell death
index (CDI) was calculated based on the cytogram by the following equation (equation 1):
(A1+A2+A4)
CDI = × 100 (5)
A3
The CDI is the ratio of total damaged cells to viable cells and is used to remove inter-
experimental variations in cell density. The net cell damage ( ΔCDI ) is derived by
subtracting the CDI of incubated control cells (Figure 3B) from that of treated cells (Figure
3C) (equation 4-2).
ΔCDIHP - ΔCDISample
% Inhibition of cell death = × 100 (7)
ΔCDIHP
where ΔCDIHP and ΔCDISample are the net cell damage caused by H2O2 and test sample,
respectively. The EC50 values were calculated from the dose-response relationship between
the concentrations of antioxidant and % inhibition of cell death.
Calculation of Log P
The log P of flavonoids was obtained by using ClogP in the Chem3D Ultra 2008
molecular package.
68 Jingli Zhang and Margot A. Skinner
Hydrogen peroxide is known to be able to induce both apoptosis and necrosis in cells
(Antunes and Cadenas, 2001; Barbouti et al., 2002; Kim et al., 2000), with the required
concentrations and exposure time dependent on the cell type being investigated. The response
of cultured human cells to H2O2 in terms of both concentration and exposure time was
determined to calculate the dosage required to kill approximately half the cells. The CDI
increased with increasing concentrations of H2O2 on HT-29 cells (Figure 4).
Figure 4. Cell death responses of cultured human HT-29 cells exposed to increasing concentrations of
hydrogen peroxide (H2O2). HT-29 cells (5 x 105 cells per ml) were exposed to different concentrations
of H2O2 and incubated at 37°C in humidified air with 5% CO2 for 24 h. Bars indicate standard deviation
from the mean of two separate determinations.
A concentration of 150 μM H2O2 was selected for the cytoprotection assay using HT-29
cells (CDI of 63.5 ± 2.7) with a 24-hour exposure to H2O2. These conditions were used in this
assay to investigate the protective effects of antioxidants against H2O2-induced total cell
death. Although H2O2 itself is a relatively unreactive species and easily scavenged by cellular
catalase (Gille and Joenje, 1992), it can cause membrane damage by increasing the release of
arachidonic acid from the cell membrane, which may account for the prolonged damage
caused by H2O2 even after being scavenged (Cantoni et al., 1989). Thus, even at low
concentrations, H2O2 can cause damage to cultured cells. These facts demonstrate that the
cytoprotection assay can be used to screen for and compare the protective effects of
flavonoids in a biologically relevant cellular environment.
Cytoprotective Activity of Flavonoids in Relation… 69
Cytotoxicity of Flavonoids
The cytotoxicities of Trolox, catechol, pyrogallol and selected flavonoids were tested on
cultured human HT-29 cells at different concentrations for 24 h using the WST-1 assay (data
not shown). None of the compounds tested affected the viability of HT-29 cells within the
concentration range used (0–20.0 μM) in this study.
Figure 5. The concentration-response curve of Trolox for protection of HT-29 cells from 150 μM
hydrogen peroxide. HT-29 cells (5 x 105 cells per well) were incubated at 37°C in humidified air with
5% CO2 for 24 h. After incubation, cells were stained with both Annexin V-FITC and PI and analyzed
by flow cytometry. Bars indicate standard deviation from the mean of two separate determinations.
Table 1. Calculated differences of heat of formation (ΔHf) between the parent flavonoid,
catechol, and pyrogallol and each possible corresponding relative radical, the
lipophilities (log P) and their cytoprotective activities
Figure 6. The influences of hydroxylation in the B ring on the cytoprotective activity of the flavones.
OH
OH OH
HO O HO O
HO O
OH OH
OH
OH O OH O
OH O
OH
OH
OH
HO O OH
HO O OH
OH
OH
OH O
OH O
4.47 ± 0.53 µM
9.98 ± 1.07 µM
Morin Myricetin
Figure 7. The influences of hydroxylation in the B ring on the cytoprotective activity of the flavanols.
72 Jingli Zhang and Margot A. Skinner
As the number of hydroxyl groups increases the EC50 value decreases except for
quercetin, which is more active than myricetin. With morin, the dihydroxyl groups in the B-
ring are arranged meta to each other. This significantly reduces its cytoprotective activity
compared with quercetin (the EC50 value of morin was four times than that of quercetin). This
result confirms that the presence of two adjacent hydroxyl groups in the B-ring plays a
significant role in the high cytoprotective activity of flavonoids. Possibly, the two adjacent
hydroxyl groups at position 3′ and 4′ in quercetin are more vulnerable to loss of a proton than
the two hydroxyl groups at position 3′ and 5′ in morin. Myricetin, which possesses ortho-
trihydroxyl (pyrogallol) groups in the B-ring, is much less active than quercetin. This
suggests that the additional 5′-hydroxyl group has a negative impact on its cytoprotective
activity. However, the cytoprotective activity of pyrogallol is much more active than that of
catechol as illustrated in Figure 8. This may be the result of the rest of the quercetin structure
(C- and A-rings) stabilizing the oxidation product (o-quninoe) as shown in Figure 9.
Figure 10. The influences of hydroxylation and methoxylation in the B-ring on the cytoprotective
activity of the flavanones.
74 Jingli Zhang and Margot A. Skinner
OH OH
OH OH OH
OH O OH O OH O
OH
OH OH OH
OH OH OH
Figure 11. The influences of hydroxylation in the B-ring on the cytoprotective activity of the
anthocyanins.
In acidic or neutral media, four anthocyanin structures exist in equilibrium (Figure 12):
the flavylium cation, the quinonoidal base, the carbinal pseudobase, and the chalcone
(Borkowski et al., 2005; Brouillard, 1983).
OH
OH
O O
OH
OH OH OH
O OH
HO O HO O
OH OH
OH O
Quinonoidal anhydro-bases
OH OH
OH OH
OH
OH O OH O
OH OH
OH OH
OH OH
OH OH
cis-Chalcone trans-Chalcone
2,3-double bond in the C-ring and the ortho-hydroxyl groups in the B-ring have positive
effect on cytoprotective activity as demonstrated by the comparison of apigenin and luteolin.
OH
OH OH
HO O HO O
HO O
OH O OH O
OH O
HO O HO O
HO O
OH OH
OH
OH O OH O
OH O
OH OH
OH O OH O OH
OH O
OH
OH
5.48 ± 0.53 µM
Cyanidin
Figure 13. Structure-cytoprotective activity comparisons of the 3-OH, 2,3-double bond and 4-keto
group of flavonoids.
However, the presence of a 2,3-double bond when the 3-hydroxyl group is absent
(apigenin and luteolin) does not significantly change the cytoprotective activity of flavonoids
relative to those that do not contain this double bond (naringenin and taxifolin). When the 3-
hydroxyl group is present (quercetin), it significantly enhances cytoprotective activity
compared with those that do not contain this double bond (taxifolin). The loss of the 4-keto
Cytoprotective Activity of Flavonoids in Relation… 77
group at the C-ring and introduction of a positive charge decreases cytoprotective activities as
seen in cyanidin and quercetin. As shown in Figure 13, quercetin, catechin and cyanidin have
identical A- and B-rings, but quercetin is more than twice as cytoprotective as catechin and
cyanidin. This observation indicates the important contribution of the 2,3-double bond and 4-
keto group to the cytoprotective activity.
OH
OH
OH
OH OH O
OH OH O
O
OH
O
OH O OH
O
HO O
OH O OH CH3 O O
O OH
OH HO OH
OH HO OH
OH O
OH OH
HO O
OH O
4.05 ± 0.48 µM
Luteolin
Similar effects are observed when cyanidin is compared with its 3-glucoside, idaein and
its 3-rutinoside, keracyanin, and when pelargonidin is compared with its 3-glucoside,
callistephin (Figure 15).
Comparison of naringenin with naringin shows that glycosylation of the 7-hydroxyl
group in a structure with a saturated heterocyclic C-ring and with a single hydroxyl group on
the B-ring has a significant negative impact on the EC50 values. Similar trends are observed
with hesperitin when a 4′-hydroxyl group in the B-ring is replaced by a methoxy and 3′-
hydroxyl group, in contrast to naringenin, compared with its rhamnoside, hesperidin, which
has a glycosylated 7-hydroxyl group. However, hesperidin is much more cytoprotective than
that of naringin because of its B-ring configuration (Figure 16).
The results presented in Figure 16 and 17 demonstrate that the presence of both 3- and 5-
hydroxyl groups is also necessary to maximize cytoprotective activity of flavonoids.
The sugar moiety is reported to have a negative effect on the oxidizability of flavonoid
glycosides (Hedrickson et al., 1994). The oxidation rate of compounds decreased as the
substituent at the 3-position became a poorer leaving group. Disaccharides are a poorer
leaving group than monosaccharides, thus rutin is less oxidizable than isoquercetin (Hopia
and Heinonen, 1999). This observation may explain why rutin displays a lower cytoprotective
activity than quercetin and isoquercetin.
Cytoprotective Activity of Flavonoids in Relation… 79
The structural criteria for the very high cytoprotective activity by flavonoids can be
summarized as: 1) the o-dihydroxy (catechol) structure in the B-ring; 2) the 2,3-double bond
in conjugation with the 4-keto group in the C-ring; and 3) the 3-hydroxyl group in the C-ring.
Thus, quercetin, for example, satisfies all the above-mentioned determinants and has the
highest cytoprotective activities among 24 flavonoids tested.
80 Jingli Zhang and Margot A. Skinner
Figure 18. Correlation between EC50 (cytoprotective activity of flavonoids) and ΔHf (the lowest heat of
formation of the ArO-H bond of flavonoids from the A- or B-ring hydroxyl group) (r2 = 0.85, n = 26).
As illustrated in Figure 18, a correlation was demonstrated between the calculated ΔHf
(the lowest differences in the enthalpy between each flavonoid’s parent compound and its
radical) values and the experimentally determined cytoprotective activity of flavonoids. There
is a strong linear correlation between the lowest ΔHf and the EC50 values and from the
regression analysis a correlation with r2 = 0.85 (n = 26) was obtained for the following
equation 8:
These findings suggest that a relatively low O-H bond dissociation enthalpy (BDE,
approximation by the lowest ΔHf), which facilitates the H-abstraction reaction between
flavonoids and reactive oxygen species (ROS) and other hydroxyl groups may well have
contributed to this reaction in the consequent steps. However, substitution of the hydroxyl
group at the C3 position of quercetin by the monosaccharide glucose in isoquercetin and
disaccharide rutinose in rutin decreases the lowest ΔHf values, but this does not result in an
increase in the cytoprotective activity. This is probably due to the fact that glycosylation
decreases the lipophilicity, and to the loss of the free hydroxyl group at the 3-position of the
C-ring. An appropriate solubility, which improves the mobility of the antioxidant across cell
membranes, is another important factor in explaining the cytoprotective effects of flavonoids.
82 Jingli Zhang and Margot A. Skinner
Correlation between Partition Coefficient (Log P) and Cytoprotective Activity (EC 50) of
Flavonoids
As shown in Table 1, flavonoids with log P values that were high (log P > 3.0) or low
(log P <1.0) had low cytoprotective activity, indicating that the cytoprotective activity of
flavonoids is associated with their affinity and distribution in lipid membranes. This is
presumably because a) at high values of log P, the flavonoid is dispersed in a lipid phase and
not located at the lipid-water interface and, b) at low values of log P, the flavonoid is located
in an aqueous phase and has insufficient solubility in the lipid phase. This can be important in
terms of paracellular transport of flavonoids and the ability to enter the cell to participate in
intracellular protection from oxidative damage. It has long been recognized that for a
chemical to be biologically active, it must first be transported from its site of administration to
its site of action and then it must bind to or react with its receptor or target, i.e. biological
activity is a function of partitioning and reactivity (Barratt, 1998). It should be noted that the
effect of membrane partitioning is not necessarily a direct relationship with lipophilicity.
Beyeler and coworkers (Beyeler et al., 1988), for example, reported that the effects of
cianidanols on rat hepatic monooxygenase increased with lipophilicity, reached a plateau,
decreased and then leveled off for the most lipophilic compounds.
As illustrated in Figure 19, for the 26 compounds tested, no correlation could be found
between EC50 and log P (equation 9).
Figure 19. Correlation between EC50 (cytoprotective activity of flavonoids) and log P (calculated by
CLogP program) of flavonoids (r2 = 0.15, n = 26).
Cytoprotective Activity of Flavonoids in Relation… 83
Figure 20. Correlation between EC50 (cytoprotective activity of flavonoids) and log P (calculated by
CLogP program) of flavonoid aglycones (without sugar substitution) (r2 = 0.51, n = 18).
EC50 = -0.45 (± 0.33) log P + 0.25 (± 0.02) ΔHf – 25.75 (± 3.04) [11]
The use of the new parameter (ΔHf ) increased the correlation coefficient r2-value from
0.15 to 0.86. The significant increase in correlation coefficient upon the introduction of ΔHf
confirmed the importance of O-H bond strength (bond dissociation energy approximated by
ΔHf), which contributed most to the model. However, log P gave a negative contribution to
the EC50 values in this model.
A QSAR model could also be derived by the introduction of ΔHf to equation 9 for
flavonoid aglycones through step-wise regression as shown in equation 12.
EC50 = 0.38 (± 0.65) log P + 0.24 (± 0.03) ΔHf – 26.25 (± 4.04) [12]
Equation 12 indicated that log P gave a positive contribution to the EC50 values of
flavonoid aglycones.
In conclusion, a QSAR model was derived from the cytoprotective activity and calculated
theoretical parameters (enthalpy of hemolytic O-H bond cleavage ΔHf and the partition
coefficient). It demonstrated that the H-abstraction was not the sole mechanism responsible
for the cytoprotective activity of flavonoids. It seems that the relative contribution of
lipophilicity (log P) is much smaller than that of ΔHf. These results demonstrated that it is
feasible to estimate the cytoprotective activities of a flavonoid from the lipophilicity and the
difference of heat of formations by using equation 11. The lipophilicity and heat of formation
Cytoprotective Activity of Flavonoids in Relation… 85
can be calculated purely by computer programs. Therefore, the QSAR model derived here
could be useful in the selection of natural flavonoids with potential cytoprotective effects.
CONCLUSION
In summary, the cytoprotection assay provides information regarding cellular activity of
antioxidants, which is important to our understanding of this area of antioxidant research.
Traditionally, the antioxidant activity of phytochemicals has been measured using a range of
chemically-defined laboratory-based assays. A cytoprotection assay that is a more
biologically relevant method than the chemical antioxidant assays has been developed and
can be adapted for use in other cell lines appropriate to tissues of interest. Using the
cytoprotection assay, the effects of compounds on cells is determined, providing information
regarding the cellular response to antioxidants, taking into account some aspects of uptake,
metabolism, location of antioxidant compounds within cells and intracellular effects on
signalling pathways and enzyme activity.
In the present study we showed that with a cell-based bioassay it is possible to identify
natural-occurring flavonoids that are gastroprotective in a model of oxidant injury. By
directly evaluating the effects of different classes of flavonoids using a lower dose of H2O2
and more chronic exposure, we showed that removal of excess ROS or suppression of their
generation by flavonoids may be effective in preventing oxidative cell death.
In this study, we carried out a theoretical investigation into the possible mechanisms
governing the cytoprotective activity of 24 different subclasses of flavonoids by
computational chemistry, and explored the correlation between experimentally determined
cytoprotective activities and physicochemical properties. It is reasonable to conclude that
multiple mechanisms regulate the protective actions of flavonoid compounds although they
contribute to the cytoprotective activity to different degrees. The cytoprotective activities of
flavonoids were strongly correlated to their calculated enthalpy of hemolytic O-H bond
cleavage (ΔHf) but weakly correlated to their lipophilicity. It is concluded that the relative
contribution of lipophilicity is much smaller than that of ΔHf to their cytoprotective capacity.
However, the balance of lipophilicity and lipophobicity is still critical in determining their
abilities to protect human cells from oxidative damage.
Judging from the improvement in the correlation coefficient in the stepwise multiple-
linear regression, we can conclude that the more precise the mechanistic information included
in the QSAR model, the better the coefficient of relation that is obtained. It is reasonable to
conclude that multiple mechanisms regulate the cytoprotection actions of flavonoids although
they contribute to cytoprotective activity to different degrees. These results suggest the
possibility of predicting the degree of contribution of different physicochemical factors
among flavonoids by their in vitro actions against oxidative stress-induced cellular damage.
86 Jingli Zhang and Margot A. Skinner
ACKNOWLEDGMENTS
This work was funded by the Foundation for Research Science and Technology Wellness
Food Programme Contract C06X0405. We thank Dr. Tony McGhie and Dr. Jeffery
Greenwood for critically reviewing the manuscript.
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Cytoprotective Activity of Flavonoids in Relation… 95
Chapter 3
A.Pizzi
ENSTIB-LERMAB, University Henry Poincare – Nancy 1,
Epinal, France
ABSTRACT
The determination by Matrix-Assisted Laser Desorption/Ionization time–of-flight
(MALDI-TOF) mass spectroscopy of the oligomeric nature of the two major industrial
polyflvonoid tannins which exist, namely mimosa and quebracho tannins, and some of
their modified derivatives indicates that: (i) mimosa tannin is predominantly composed of
prorobinetinidins while quebracho is predominantly composed of profisetinidins, that (ii)
mimosa tannin is heavily branched due to the presence of considerable proportions of
"angular" units in its structure while quebracho tannin is almost completely linear. These
structural differences also contribute to the considerable differences in viscoity of water
solutions of the two tannins. (iii) the interflavonoid link is more easily hydrolysable, and
does appear to sometime hydrolyse in quebracho tannin and profisetinidins, partly due to
the linear structure of this tannin, and confirming NMR findings that this tannin is subject
to polymerisation/depolymerisation equilibria. This tannin hydrolysis does not appear to
occur in mimosa tannin in which the interflavonoid link is completely stable to
hydrolysis. (iv) Sulphitation has been shown to influence the detachment of catechol B-
rings much more than pyrogallol-type B-rings. (vi) The distribution of tannin oligomers,
and the tannins number average degree of polymerisation obtained by MALDI-TOF, up
to nonamers and decamers, appear to compare well with the results obtained by other
techniques. As regards procyanidin tannins, it has been possible to determine for
mangrove polyflavonoid tannins that: (i) procyanidins oligomers formed by
catechin/epicatechin, epigallocatechin and epicatechin gallate monomers are present in
great proportions. (ii) oligomers, up to nonamers, in which the repeating unit at 528-529
Da is a catechin gallate dimer that has lost both the gallic acid residues and an hydroxy
group are the predominant species. (iii) oligomers of the two types covalently linked to
each other also occur.
98 A.Pizzi
INTRODUCTION
Polyflavonoids also called condensed tannins are natural polyphenolic materials.
Industrial polyflavonoid tannin extracts are mostly composed of flavan-3-ols repeating units,
and smaller fractions of polysaccharides and simple sugars. Two types of phenolic rings
having different reactivities are present on each flavan-3-ol repeating unit, namely A-rings
and B-rings, with each repeating unit being linked 4,6 or 4,8 with the units which precede and
follow it.
Oligomeric Nature, Colloidal State, Rheology… 99
(OH)
OH
HO
8
O 1' B
7A 2 OH
3
6 5 4
OH
(OH)
Recently, the radical and ionic mechanisms of the reaction of autocondensation and
networking of polyflavonoid tannins induced by bases and by weak Lewis acids has been
described [2-8]. Different polyflavonoid tannins however present different structures and
different average molecular masses, and as a consequence often present peculiarly different
behaviour in their application [10]. The most common method of examination of the relative
structures of polyflavonoid tannins, and of their differences, is by 13C NMR [10].
form the oligomers, but certain units, in the minority are also linked C4 to C8. Quebracho
tannin and Mimosa tannin are the two main exponents of this class. Quebracho gave clear
spectra showing the degree of polymerization of the building units and oligomer series with
masses of the repeat units of 272 Da and 288 Da (Figure 1a; Table 1). For each oligomer,
substructures with mass increments of 16 Da appear, indicating different combinations of
various substructures. As quebracho is mainly based on combinations of resorcinol, catechol
and pyrogallol building blocks the following monoflavonoids and their oligomers can be
expected to be present:
The masses of units A and B are 274 Da and 290 Da respectively. Combinations of these
masses can be used to calculate the masses of the oligomer peaks in the spectra according to
the expression M+Na+ = 23(Na) + 2 (endgroups, 2xH) + 272A +288B (Table 1). As can be
seen in the spectra, there are more peak series which are due to different endgroups. They
have the same repeat units, for example 683-956 Da and 1555-1827 Da in Figure 1. The peak
at 683 is very near to a result of 688 Da which would be obtained by the loss of both a B-ring
plus the three-carbons chain from the heterocycle of the lower terminal repeat unit, be this of
type B or of type A, to yield two flavonoid units linked to a resorcinol phenoxy anion.
The peak at 585 Da is also explained by the presence of a dimer according to the same
equation above composed of a A-unit plus a B-unit plus 2 H endgroups plus Na+. The peak at
375 Da is obtained from the 585 Da dimer by elimination of a catecholic B-ring (585-110 =
375).
Oligomeric Nature, Colloidal State, Rheology… 101
There is however an alternate, and more correct explanation for the 683 Da peak.
Industrial quebracho tannin extract is sulphited/bisulphited, which introduces a sulphite or
sodium sulphite group on the C1 of the flavonoid structure and causes the opening of the
heterocycle ring. Thus, if one of the flavonoid units of a 857 Da trimer loses its catechol B-
ring (-110) from a type A repeat unit as well as the –SO2- group (-64), the 683 Da signal is
obtained.
Table 1. MALDI fragmentation peaks for industrial quebracho tannin extract. Note that
the predominant repeat units in this tannin is 272 Da, indicating that this tannin is
predominantly a profisetinidin
Figure 1. MALDI mass spectrum of (A) natural sulphited quebracho tannin extract. (B) details of the
600-1300 Da range with indication of the relevant 272 Da repeat unit. (C) details of the 1300-2000 Da
range with indication of the relevant 272 Da repeat unit.
Oligomeric Nature, Colloidal State, Rheology… 103
This is more likely as the introduction of the –SO2- group should favour under certain
conditions the elimination of the B-ring of the unit. The origin of the much smaller 665 Da
peak is the same but by elimination of the –SO2H group (-65) and of a pyrogallol B-ring (-
126) from a type B repeat unit. The fact that the intensity of the 665 Da peak is considerably
lower than that of the 683 Da peak indicates the novel finding that as a consequence of
sulphitation catechol rings appear to be much more easily detached than pyrogallol ones from
a flavonoid unit. That the 683 peak is caused by the presence of the sulphonic group on C1
and the relative ease of decomposition indicated above is shown by the fact that in mimosa
tannin which in general is not sulphited the 683 Da peak does not exist while presenting a
very small peak at 687 Da which come from the first explanation (Figure 2a).
Also of interest are the existence of peaks at 1965, 2237, 2510 and 2800 Da for
commercial quebracho tannin, these representing respectively heptamers, octamers, nonamers
and decamers (Figure 1). Tannins are not easily water soluble at this higher molecular weight
and thus it is of interest to find definite proof of the existence of such higher molecular weight
oligomers in a commercial tannin extract. The sample in question had been found by 13C
NMR to have a number average degree of polymerization of 6.74 [19,20] which appears to
confirm the existence of such higher molar mass oligomers in this commercial tannin extract.
The same type of pattern is obtained for solvent purified commercial quebracho extract
(MALDI spectrum not reported here), in which all the carbohydrates have been eliminated,
confirming that the patterns observed are really due to the polyflavonoid components of the
tannin extract. It is, however, of interest to note that the tannin extract which has undergone
an acid/base treatment [18] to obtain an adhesive intermediate gives at best a pentamer at
1967 Da, see Figure 2. This is accompanied by a considerable increase in the proportion of
the 858 Da trimers, of the 727 degraded trimers (2 flavonoid units + 1 A-ring + its C4), of
degradation product composed of a single flavonoid unit linked to a single A-ring of another
flavonoid unit (375 da) and also an increase in the 1130 Da tetramers confirming that the
treatment to yield a tannin adhesive intermediate clearly induces some level of hydrolysis of
the interflavonoid bond and hence some level of depolymerization in quebracho tannin.
104 A.Pizzi
Figure 2. MALDI mass spectrum of (a) acid/base treated modified quebracho tannin extract. (b) details
of the 600-1300 Da range, (c) details of the 1300-2000 Da range.
Oligomeric Nature, Colloidal State, Rheology… 105
This confirms previous findings [6-8,18] obtained by 13C NMR that contrary to what
widely thought the interflavonoid bond in the profisetinidins/prorobinetinidins of quebracho
tannin are fairly labile and that this particular type of tannin can be subject to some
depolymerization. It also confirms what has been up to now only a suspicion, namely that the
decrease in viscosity [18] of tannin solutions as a consequence of acid/base treatments is not
only due to hydrolysis of the hydrocolloid polymeric carbohydrates present in the extract, but
also to the decrease in degree of polymerization of the tannin itself, this at least in the case of
quebracho tannin. It is also interesting to observe a definite, clear peak at 605 Da (see Figure
2) which can only belong to a pure robinetinidin dimer (289+289+25 = 605), MALDI-TOF
analysis appearing to indicate here that it is the interflavonoid inter-fisetinidin link, or at least
links in which fisetinidin units are involved which appear to be more sensitive to cleavage.
The acid/base treatment to produce a tannin adhesive intermediate involves the use of acetic
anhydride or maleic anhydride for the acid hydrolysis phase. As the treatment is done in water
solution but being the tannin extract strongly colloidal in nature a question of interest is to
know if some of the tannin –OH groups have been acetylated within the micelles present in
the solution and before the induced hydrolysis has drastically decreased the level of
colloidality of the system. Past investigations by 13C NMR and by other techniques [18]
indicate that a certain amount (small) of acetylation appears also to occur, this being of
importance in accelerating subsequently, on application, the polycondensation of tannins with
aldehydes. MALDI-TOF appears to confirm this by the presence of small but detected peaks
at 772 Da (in theory 769) and 902 Da (see Figure 2), respectively a flavonoid dimer and a
flavonoid trimer both monoacetylated.
The MALDI-TOF analysis of mimosa tannin extract (Figure 3) indicates the presence in
the tannin of oligomers to the maximum of octamers (2333 Da) in line with the lower number
average degree of polymerzation of 4.90 obtained by other means for this tannin [19,20], and
the distribution obtained is shown in Table 2. The flavonoid repeating units present in this
tannin extract are of type A and B as for quebracho but with a relatively important proportion
of units of type C.
The correct equation to calculate the different possibilities does then become M+Na+ =
23(Na) + 2 (endgroups, 2xH) + 272A + 288B + 304C (Table 2). Table 2 indicates that many
valid combinations of different repeating units are possible. There are however some cases in
which unequivocal assignement of the structure can indeed be done.
106 A.Pizzi
Table 2. MALDI fragmentation peaks for industrial mimosa tannin extract. Note that
the predominant repeat units in this tannin is 288 Da, indicating that this tannin is
predominantly a prorobinetinidin
This is the case of angular tannins, namely oligomers in which a repeating unit of type C
is bound through both its 6 and its 8 A-ring sites to A and B type units, with its C4 sites
equally bound and unbound.
Oligomeric Nature, Colloidal State, Rheology… 107
Figure 3. MALDI mass spectrum of (a) natural mimosa tannin extract. (b) details of the 600-1300 Da
range with indication of the relevant 288 Da repeat unit. (c) details of the 1300-2000 Da range.
Oligomeric Nature, Colloidal State, Rheology… 109
A further interesting difference between mimosa and quebracho tannins can be observed
by comparing the results in Figures 1 and 3 and Tables 1 and 2. In quebracho the predominant
repeat unit has 272 Da (a type A unit), while in mimosa the predominant repeating unit is of
288 Da (a type B unit). This is particularly evident in the higher oligomers for the two
tannins. Based on this, on the dominant fragments for different oligomers and on the relative
intensities for the different peaks in Figures 1 and 3 it is possible to conclude that quebracho
tannin is composed of between 20% and 30% of B units and of between 70% and 80% of A-
type units. Quebracho is then predominantly a profisetinidin. Mimosa tannin instead is
composed predominantly of between 50% and 70% of type B units and only of between 15%
and 25% of type A units. Mimosa tannin is then predominantly a prorobinetinidin. It is also
interesting to note that the number average degree of polymerization obtained from the
MALDI-derived oligomer distributions yield values of 6.25 and 5.4 for quebracho and
mimosa tannins respectively. Considering the variability of such natural materials, these
values compare well with the DPn values of 6.74 and 4.9 for the same tannins obtained by 13C
NMR and other techniques [19,20].
It must be born in mind that in some mainly prorobinetinidin/profisetinidin tannins the
structure of the oligomers present is not fixed and immutable. There are almost pure
procyanidin oligomers present mixed with pure prorobitenidin oligomers, mixed with hybrid
prorobinetinidin/profisetinidin oligomers and with hybrid prorobinetinidin/
profisetinidin/procyanidin oligomers. An example of this is the case of the flavonoids from
the bark of Acacia mangium, a species now extensively cultivated in Brazil and in South East
Asia (Figure 4). This tannin presents two main "mixed" patterns of oligomers overimposed
one on the other. As an example (Figure 4):
(1) Starting from the 1210 peak if one adds 289 Da the series 1210-1499-1788-2076.7-
2365.4 etc up to the 3535 peak (that is very small) is observed in Figure 4, thus a
pure prorobinetinidin pattern of trimers, tetramers, pentamers etc., up to oligomers
comprised of 13 repeating units (although their proportion is quite low). This
indicates that pure prorobinetidin oligomers , thus pure B type units linked together
with perhaps one or two C units but without any A units linked to them do indeed
exist.
(2) Starting again from the 1210 Da peak also a pattern adding 272-273 Da is present,
namely 1210 - 1483 - 1756 Da but this stops there and the intensity of the peaks is
lower, thus they are less abundant. This is hence a mixed
prorobinetinidin/profisetinidin series of oligomers. The fisetinidin repeating unit is
less abundant, as also indicated by the intensity of these peaks. Adding a 289 Da B
unit then one passes to the 2044.9 mass oligomer. Another 289 unit will bring to the
2334 oligomer. Equally, starting from the 1499 peak adding 272-273 Da one also
gets the sequence 1499 - 1772 - 2044.9 and again it stops there. Adding a 289 Da
unit to the 2044.9 peak and oligomer one gets the peak at 2334 Da, indicating clearly
that fisetinidins and robinetinidins mixed oligomers, where both units of type A and
B are linked together exist (still with a majority of B units). It also indicates that
some peaks such as the 2044.9 Da, for example ,are the superposition of two
different types of oligomers.
(3) In Figure 4 there is also a very interesting, clearly identifiable, very low intensity
pattern 752 - 1041 - 1330 - 1635 - 1924 - 2217 - 2506 where all the peaks are
110 A.Pizzi
separated by a 288-290 repeating motive except for the 1330 - 1635 one that is
separated by a 304 Da C unit. This is a definite series of angular oligomers up to at
least a 7 repeating units one. Thus, a series of angular tannins of 3+1, 3+2 and 3+3
robinetinidin units linked to a branching C unit. (an angular tannin means there is a C
branching unit). It is angular if there are two flavonoid units linked both to the A ring
of the C unit, it is branched if on top of this there is another flavonoid series attached
to the C4 of the C unit. The pattern indicated is almost certainly a series of angular or
branched oligomers of a length never determined before. By the way, whenever there
is a C unit linked to A and B units one could have an angular tannin. However in
cases where several C-units linked to each other are present, then one has definetely
a prodelphinidin or procyanidin oligomer. Furthermore the 289 Da unit can equally
be a prorobinetinidin but also a catechin unit, hence one could also have angular
tannins with the 289 B unit if one of the -OHs is situated on the A ring rather than the
B-ring.
%Int. 144 mV[sum= 28296 mV] Profiles 1-197 Smooth Av 50 -Baseline 100
1499.1
1210.1
100
90
80
1788.0
1483.1
1194.2
70
921.0
60
1772.0
50
905.2
2076.7
40
1804.0
1515.2
2060.6
30
2365.4
667.8
1178.1
1756.0
2653.8
20
1330.4
2044.9
1635.1
752.8
2942.4
1924.3
1041.9
2334.2
934.8
3246.5
2212.7
576.1
10
0
500 1000 1500 2000 2500 3000 3500 40001[c].O
Mass/Charge
For Rhizophora apiculata mangrove tannins, the MALDI –TOF spectra in Figure 5a,b,c
indicates clearly that alternate repeating units with mass increments of 264-264.9 Da occur.
These have not been identified by previous analysis by other methods [23-26] in mangrove
tannins indicating possibly the presence also of other monomers than those shown in Figure 1
and/or different combinations of various structures. Combination of the masses of the
catechinin monomers shown in Figure 5 can be used to calculate the masses of the oligomer
peaks in the spectra according to the expression M+Na+ = 23(Na) + 2 (endgroups, 2xH) +
290.3 (-2H)A + 306.3 (-2H)B + 442.4 (-2H)C (Table 3). The only problem about this is the
presence of a repeating structure the MW of which is regular at 264.0 – 264.9 Da.
1892.6
100
1231.7
90
1495.9
2157.4
1760.1
80
70
1666.3
2024.7
1345.6
1814.7
60
2288.8
1099.6
2422.3
2193.0
1725.2
1976.7
1215.3
50
2557.3
967.3
1507.5
2643.1
1303.5
2817.2
40
1073.4
2947.6
3081.1
3169.1
3423.0
30
927.0
20
10
0
500 1000 1500 2000 2500 3000 3500 1[c].N
Mass/Charge
Figure 5. (Continued).
112 A.Pizzi
%Int.
100
264
264 264
90
80 264 264
264
264
70
264
60 264
264
50
264
40
30
20
10
0 1[c].N2
1628.2
1363.6
100
1231.7
90
1495.9
1760.1
80
70
1666.3
1649.7
1345.6
1377.5
60
1725.2
1681.6
1524.7
1698.0
1783.8
1215.3
50
1577.6
1553.0
1507.5
1741.4
1594.8
1303.5
1454.5
1328.5
1421.8
40
1487.9
1200.0
1612.5
1248.3
1394.9
1661.7
30
20
10
0
1[c].N
Figure 5. MALDI mass spectrum of (a) mixed Rhizophora apiculata mangrove tannin extract, 500-
3900 Da range. (b) indication of the relevant 264 Da repeat unit.. (c) details of the 1120-1800 Da range.
This unit has not been identified before, and we will call it here structure D. Calculation
of the MALDI masses indicate that certain peaks can only be explained by the presence of
epicatechin gallate units in which the gallic acid residue has being removed of 274.3 Da
(structure E), these being related to the unknown structure. The equation than becomes
M+Na+ = 23(Na) + 2 (endgroups, 2xH) + 290.3 (-2H)A + 306.3 (-2H)B + 442.4 (-2H)C +
274.3 (-2H)E. In Table 3 are shown the results of the combination of monomer units forming
the different oligomers observed by MALDI-TOF. It must be noticed that only very few of
the dominant peaks (Figure 5a) can be explained only on the basis of the catechinic structures
A, B and C. Some mass peaks however are not easily explained without the use of structure
D. The majority of the dominant peaks are shown in Tables 4 and 5.
Oligomeric Nature, Colloidal State, Rheology… 113
Table 3. MALDI fragmentation peaks for mixed Rhizophora spp mangrove tannin
extract. Note that the predominant repeat unit in this tannin indicate that it is
predominantly a procyanidin
Table 4. MALDI fragmentation peaks for mixed Rhizophora spp mangrove tannin
extract. Note that the predominant repeat units in this tannin is 528-530 Da, indicating
that in its main series of peaks this tannin may present pure profisetinidin oligomers as
predominant components
*Dominant fragment.
Table 5. MALDI fragmentation peaks for mixed Rhizophora spp mangrove tannin
extract. Note that the predominant repeat units in this tannin is still 528-530 Da,
indicating that in this MALDI series of peaks this tannin may present predominantly a
profisetinidin component but linked to procyanidins units too
*Dominant fragment.
Oligomeric Nature, Colloidal State, Rheology… 115
Thus, in Table 4 is shown the main series of dominant MALDI masses indicating that
oligomers of this unit appear to occur in apiculata mangrove tannins. This is the most likely
case seeing the regular progression from trimer to octamer in Table 4.Thus, mixed oligomers
where a procyanidin oligomer formed by structures of type A, B and C (1363.6 Da) is linked
to progressively increasing number of D structure oligomers are possible. The results in Table
3 of the second more important series of recurrent MALDI peaks clearly confirms that mixed
procyanidin and D oligomers covalently linked do exist in such mangrove tannins because
none of the masses of the series 835 Da, 1099 Da, 1363 Da, 1628 Da, 1892 Da, 2157 Da,
2422 Da can be explain without having units of structures A, B and C linked to the D
oligomers. It appears most likely then that both pure oligomers of the two types as well as
linked mixed oligomers do coexist in this tannin.
A mainly prodelphinidin tannin, namely pecan nut tannin extract was also examined
(Figure 6) [33]. The main prodelphinidin repeat unit has a molar mass of 306 Da and the main
fragments found arrived only up to trimers (304+304+288+2+23 = 921 Da). This unusual
result leads to two consequences. In pecan nut tannin extract robinetinidin units are linked
within the prodelphinidin main oligomers, and that the interflavonoid bond of prodelphinidins
is particularly prone to cleavage (this is known to be so). It means that the finding of trimers
only , when the number average degree of polymeryzation of this tannin is known to be of
5.50 [19,20] , indicates that the cleavage of the interflavonoid bond here is mainly a
fabrication of the method of analysis used and if MALDI-TOF has to be used in this case
much milder conditions needed to be used.
In conclusion MALDI-TOF is a suitable method for examining polyflavonoid tannin
oligomers and one that is able to determine through this technique facts on polyflavonoid
tannins which are already known by using other approaches. It also appears capable however
to determine aspects of the structure and characteristics of the tannins which are too difficult
to determine by other techniques.
In the present investigation of the two major commercial polyflavonoid tannins it has
been possible to determine by MALDI-TOF that: (i) mimosa tannin is predominantly
composed of prorobinetinidins while quebracho is predominantly composed of
profisetinidins, that (ii) mimosa tannin is heavily branched due to the presence of
considerable proportions of "angular" units in its structure while quebracho tannin is almost
completely linear. These structural differences also contribute to the considerable differences
in viscoity of water solutions of the two tannins. (iii) the interflavonoid link is more easily
hydrolysable, and does appear to sometime hydrolyse in quebracho tannin and profisetinidins,
partly due to the linear structure of this tannin, and confirming NMR findings that this tannin
is subject to polymerisation / depolymerisation equilibrium. This is not the case for mimosa
tannin in which the interflavonoid link is completely stable to hydrolysis. (iv) Sulphitation
has been shown to influence the detachment of catechol B-rings much more than pyrogallol-
type B-rings. (v) The distribution of tannin oligomers, and the tannins number average degree
of polymerisation obtained by MALDI-TOF appear to compare well with the results obtained
by other techniques.
Figure 7. Strain sweeps at ω = 1 rad s-1 for mimosa tannin extract solutions at different concentrations:
( ) 30%, (ο) 40%, (Δ) 50% concentration. (ο) G′ curves; (•) G″ curves.
Figure 8. Strain sweeps at ω = 1 rad s-1 for quebracho tannin extract solutions at different
concentrations: ( ) 30%, (ο) 40%, (Δ) 50% concentration. (ο) G′ curves; (•) G″ curves.
120 A.Pizzi
Figure 9. Strain sweeps at ω = 1 rad s-1 for pine tannin extract solutions at different concentrations: (♣)
20% ; ( ) 30%, (ο) 40%, (Δ) 50% concentration. (ο) G′ curves; (•) G″ curves.
Figure 10. Strain sweeps at ω = 1 rad s-1 for pecan tannin extract solutions at different concentrations:
(♣) 20% ; ( ) 30%, (ο) 40%, (Δ) 50% concentration. (ο) G′ curves; (•) G″ curves.
These figures also indicate that in the low percentage strain region both moduli appear to
be fairly independent of the applied strain amplitude as shown from the almost parallel trend
of the two moduli curves. However, when the percentage strain increases the value of G’
Oligomeric Nature, Colloidal State, Rheology… 121
starts to decrease significantly in relation to the value of G”, a trend which becomes slightly
more evident the lower is the concentration studied. Furthermore, the G### linearity limit
appears to decrease somewhat with increasing concentration. This indicates that,
notwithstanding the purely viscous liquid behaviour of these tannin extract solutions, (i)
microstructures exists for these extracts in solution , a fact supported by the known colloidal
interactions for these materials already reported [27,29-32,34-39], and (ii) that such
microstructures are labile as they are significantly broken with applied shear, leading to a
critical strain after which a significant decline in the elastic modulus results, a fact supported
by the well known thixotropic behaviour of concentrated, commercial polyflavonoid tannin
extracts water solutions [1,27,30,34,36]. For the two higher concentrations used, and also for
the 30% concentration, with only one exception, as the extracts where used at their natural pH
which is in the 4.2-5.1 range there was no case where G’ > G” and no concentration occurred
at which G’ = G”, as this behaviour has been associated with much higher alkalinity ranges
[30].
While these are general trends for all the four polyflavonoid tannins, some important
difference between the tannins also exists, mainly based on their differences in viscosity.
Firstly, G′ starts decreasing according to the different typical molecular masses of each
tannin: to a higher molecular mass corresponds a later start in the decrease of G′. The main
difference occurs for pine tannin extract solutions where two plateau for G’ and G” occurs
with this becoming more evident the higher is the tannin solution concentration. The
existence of another transition at lower percentage strain also indicates the presence of
another type of labile microstructure. Secondary forces associations between tannin oligomers
and oligomeric sugars, derived from degraded hemicelluloses which are always present in
consistant proportions in these extracts, are well known [1,19,20,30]. They have often caused
in the past the incorrect determination of absurdly high molecular masses for tannin
oligomers due to the formation of ionic polymers between tannins and carbohydrate
oligomers. This transition is particularly evident in Figure 9 for the pine tannin extract. It is
not possible to conclude from the available data if this is the cause of the additional transition
in pine tannin, or rather if the affinity of carbohydrates for tannins is the transition that occurs
for all the four tannins. The decrease of these secondary forces associations at higher
percentage strains will cause the system to appear to behave as composed of species of lower
average molecular mass with the consequent trend observed for G’ in Figures 7-10.
Figures 11-14 show the variation of G’ and G” with frequency for the four tannins each at
four different concentrations. The lower the slope of the curves the closer to a newtonian
behaviour is the behaviour of the tannin extract solution. For all the tannins and for all the
concentrations examined G’ values are smaller than G” and all G” values are relatively low
and increase progressively with increasing frequency: the tannin extracts are behaving
essentially as a viscous liquid as discussed earlier, the only exceptions being the tannin
extract solutions at 20% concentration where G’ and G” cross-over points do indeed occur.
For concentrations higher than 20% this appears to suggest that the tannin oligomers are well
separated and that, once the interactions with the carbohydrate are eliminated or minimized,
there is little possibility of molecular interaction and entanglement, even at the higher
concentration.
122 A.Pizzi
Figure 11. Elastic modulus (G′) and viscous modulus (G″) as a function of frequency (ω) for mimosa
tannin extract solutions at different concentrations: ( ) 30%, (ο) 40%, (Δ) 50% concentration. (ο) G′
curves; (•) G″ curves.
Figure 12. Elastic modulus (G′) and viscous modulus (G″) as a function of frequency (ω) for quebracho
tannin extract solutions at different concentrations: (♣) 20% ; ( ) 30%,(ο) 40%, (Δ) 50% concentration.
(ο) G′ curves; (•) G″ curves.
Oligomeric Nature, Colloidal State, Rheology… 123
Figure 13. Elastic modulus (G′) and viscous modulus (G″) as a function of frequency (ω) for pine
tannin extract solutions at different concentrations: (♣) 20% ; ( ) 30%, (ο) 40%, (Δ) 50%
concentration. (ο) G′ curves; (•) G″ curves.
Figure 14. Elastic modulus (G′) and viscous modulus (G″) as a function of frequency (ω) for pecan
tannin extract solutions at different concentrations: (♣) 20% ; ( ) 30%, (ο) 40%, (Δ) 50%
concentration. (ο) G′ curves; (•) G″ curves.
124 A.Pizzi
Thus, is there little interaction in the tannin extracts, resulting in smaller elastic
contributions, or must one consider also other parameters? In this respect there are some
differences in the curves of the four natural extracts. Considering the differences in
percentage strain which had to be used for the different tannin solutions it can be noticed that
the value of the two moduli are quite different passing from one tannin to the other (mimosa
and quebracho appear for example to have have similar G’ and G” values; in reality this is not
the case as the two figures are respectively at 100% and 10% strain respectively (Figures 11,
12). This partly reflects the level of molecular association occurring in each different extract.
However, (Figure 13) in the pine tannin extract solutions the differences in value between G’
and G”, at the same imposed frequency, are smaller than for the other three tannins, and the
value of both moduli is higher (Figure 13). This means that at parity of conditions the elastic
component of a pine tannin extract solutions is both in absolute and proportionally greater
than in the solutions of the other tannin extracts. This indicates that the determining
parameters as regards the elastic response of a tannin extract solution are both (i) the greater
average molecular mass of the tannin as well as (ii) the intensity of the tannin interaction with
the carbohydrate oligomers present in the extract, which is related to both the amount of
carbohydrate oligomers present and to their average molecular mass as well as to (i) above,
however feeble such interactions might be. The first of these two parameters would lead to
pine and quebracho tannin solutions having the higher moduli values, followed by pecan
tannin extract and mimosa tannin extract (which is indeed the case if one considers the
differences in percentage strain which had to be used, and the average molecular masses
reported above [19,20]). The second of these two parameters would place the elastic response
of the tannin as highest for pine and quebracho (the first mainly but not only for the higher
proportion of carbohydrates, and the second for their and their carbohydrates higher
molecular mass), followed by pecan (the carbohydrates content of which is very low) and last
mimosa. In Figures 11-14 pine tannins and quebracho are the ones with higher numerical
values of elastic response (if one considers the differences in percentage strain which had to
be used) and pine also presenting the higher proportional value, followed by pecan and by
mimosa.
Figures 11-14 also indicate that G’ and G” present a pronounced frequency dependence
for mimosa and quebracho tannin extracts, as indicated by the sharper slope of the moduli
curves. The same figures indicate that G’ and G” present instead little or no frequency
dependence for pine and pecan tannin extracts.
A case apart appears to be the 20% concentration case for the three tannins for which
such a concentration was also used (mimosa was not used at 20% concentration). A G’ and
G” cross-over point ( G’ = G”, and then at increasing frequency G’ > G”) occurs for all the
three tannin extracts, as it also occurs for mimosa tannin solutions at 30% concentration, in all
four cases at the extreme range of the frequency used (Figures 11-14). This again confirms
that on top of the effect of the pH [30] , the viscoelastic response of the tannin extracts
obtained in these cases is due to intermolecular associations. It is a very clear indication that
molecular associations between tannin molecules, and particularly between tannin molecules
and carbohydrates to form labile structures of greater apparent molecular mass by either
colloidal or other interactions, do indeed occur. An example of the behaviour of a solution of
a polymeric carbohydrate, such as gum arabic, at the same four concentrations used for the
tannins (Figure 15) indicates both a clear rubbery plateau at the two higher concentrations,
but also that G’ = G” cross-over points occur. At the two lower concentrations (20%-30%), at
Oligomeric Nature, Colloidal State, Rheology… 125
the extreme of the frequency range used, it is is indeed the value of G’ which becomes higher
than the value of G”, imitating what seen for the tannin extracts at the same concentration,
again confirming the partecipation of carbohydrate oligomers in imparting under certain
conditions viscoelastic behaviour to tannin extract solutions, a purified tannin without any
carbohydrates rather behaving as a viscous liquid only [37-39].
Figure 15. Elastic modulus (G′) and viscous modulus (G″) as a function of frequency (ω) for gum
arabic solutions at different concentrations: (♣) 20% ; ( ) 30%, (ο) 40%, (Δ) 50% concentration. (ο) G′
curves; (•) G″ curves.
In conclusion commercial, industrially produced mimosa, quebracho, pine and pecan nut
polyflavonoid tannin extracts water solutions of different concentrations were examined by
rheometry by measuring dynamic moduli both as a function of strain amplitude and at varying
frequency. The water solutions of these materials have been found to behave in general
mainly as viscous liquids at the concentrations which are generally used for their main
industrial applications [1,27,40]. Clear indications of viscoelastic response are also
noticeable, among these the cross-over of the elastic and viscous moduli curves at the lower
concentrations of the range investigated, with some differences being noticeable between
each tannin and the others, pine and quebracho tannin extracts showing the more marked
viscoelastic behaviour. Other than pH dependence (and related structural considerations), the
parameters which were found to be of interest as regards the noticeable viscoelastic behaviour
of the tannin extracts were the existence in the solutions of labile microstructures which can
be broken by applied shear. This is supported by the well known thixotropic behaviour of
concentrated, commercial polyflavonoid tannin extracts water solution [1,30,34,36]. Such
microstructures appear to be due or (i) to the known colloidal interactions of these materials
already reported [1,30,34,35,37-39,41], or (ii) to other types of secondary interactions [35,37-
39] between tannin oligomers and particularly between tannin and carbohydrate oligomers
126 A.Pizzi
present in the extracts. The latter is supported by the dependence of this effect from both the
average molecular masses of the tannin and of the carbohydrate oligomers.
degradation of a material, to measure the antioxidant capabilities of any surface finish could
be defined as the measurement of two different parameters. These are:
(i) The rate at which the tannin is able to form a radical; this is determined either by the
rate of radical transfer from a pre-existing radical species to the tannin to form a
more stable phenoxyl radical, or by the rate of radical formation on light irradiation
of the tannin, and
(ii) The rate of radical decay of tannin phenoxyl radicals formed.
The first parameter defines the ease and readiness of the tannin in subtracting a radical
from, for instance, the substrate; the easier and the more rapid this transfer is, the greater are
the antioxidant capabilities of the tannin. The second parameter defines how stable is the
tannin phenoxyl radical. Here two interpretations are possible: (i) in general the more stable is
the radical, hence the slower is the rate of radical decay, the more marked is the inhibition of
radical degradation of the substrate and hence the better are the antioxidant properties of the
tannin, but (ii) in the case of tannin radicals considerably more reactive than the substrate,
hence where radical termination reactions between two tannin radicals are favourite, the faster
the rate of radical decay, hence the faster the quenching between themselves of the tannin
radicals formed, the better are the antioxidant properties of the tannin. Cases (i) and (ii) define
two different effects: while case (i) has general applicability in all cases, case (ii) might
assume disproportionate importance in the case of experiments, like those reported here, in
which the substrate is not present. This discussion is then limited at evaluating the importance
of the radical decay reaction only from the point of view of case (ii): the slower the radical
decay rate, the greater is the antioxidant power of the tannin. An example of the type of
cumulative curve obtained is shown in Figures 16 and 17.
Figure 16. Intensity increase and decrease of the average of the two opposite symmetrical peaks
representing the ESR signal during UV irradiation experiments of Quebracho flavonoid tannin extract
powder without vacuum, in air, and in a quartz sample-holder. The initial part of the curve up to the
maximum of intensity describes the increase in radical concentration as a function of time during
irradiation while the decreasing intensity section describes the radical decay reaction from the moment
the UV lamp has been switched off.
128 A.Pizzi
Figure 17. Intensity increase and decrease of the average of the two symmetrical, opposite peaks
representing the ESR signal during UV irradiation experiments of Mimosa flavonoid tannin extract
powder under vacuum and in a glass sample-holder. The initial part of the curve up to the maximum of
intensity describes the increase in radical concentration as a function of time during irradiation while
the decreasing intensity section describes the radical decay reaction from the moment the UV lamp has
been switched off.
The increase in intensity of the ESR signal, which corresponds to the reaction of radical
formation due to the irradiation of the three flavonoid and one hydrolysable tannins
examined, can be modelled by a first order kinetic law of the type I/Io=aekt (Table 7).
Comparison of the rate constants or of the semitransformation times t1/2 allows a few
deductions, namely
Thus, in air, the above results (Table 7) indicate that mimosa and quebracho should
present the best antioxidant characteristics, with the results of mimosa under vacuum
indicating a greater consistency, while pecan and oak tannin appears to have poorer
antioxidant characteristics.
Equally, the decrease in intensity as a function of time of the ESR signal after stopping
the irradiation of the specimen, hence the radical decay reaction itself, is correctly described
by first order kinetics of the form I/I0=a'e-k't and shown in Table 8 and indicates that
1. In general without vacuum, oak and pecan mantain the radical for much longer than
quebracho and even longer than mimosa tannin.
Oligomeric Nature, Colloidal State, Rheology… 129
2. In general with vacuum, quebracho presents the slowest radical decay reaction,
followed by oak, then pecan and last by mimosa tannin.
3. Differences in the behaviour between quartz and glass cells are noticeable. With
vacuum for instance, pecan in a glass cell appears to have the slowest radical decay
rate, an unexpected occurrence considering all the other results.
Table 7. Radical formation (= First reaction step) kinetics according to a first order
kinetic law derived from electron spin resonance experiments. (I/Io=a ekt)
Thus, in radical formation the order of faster to slower rate remains the same with just
differences in relative rates determined by the conditions used (vacuum or not, quartz or
glass), and is
mimosa=quebracho>>pecan>/=oak
In the radical decay reaction instead the slower to faster rate changes quite considerably
according to the conditions, particularly but not only according to the presence or absence of
vacuum. Thus, without vacuum and the air singlet oxygen present the rate of radical decay is
mimosa>pecan>/=oak>>quebracho
130 A.Pizzi
In air then, the balance of the two reactions indicate that the differences between the
various tannins should not be major. As, however, radicals have to form before they can
decay, the rapidity of radical assumption or formation is the most likely important factor, and
thus mimosa and quebracho should be considerably better as antioxidants than the other two
tannins. If it is considered that quebracho is as rapid as mimosa to form radicals but that its
radicals definitely present a slower radical decay rate, the first conclusion which could be
drawn is that quebracho appears to have a slightly better overall antioxidant behaviour than
mimosa, but that such a difference is not likely to be very marked. All three flavonoid tannins
appear to have considerably better behaviour as antioxidants than the hydrolysable tannin.
The general behaviour of tannins described above can also be seen however from a point
of view of relative intensity of the ESR signals, thus total radicals formed, rather than just
from a purely kinetic rate point of view (table 8). Table 9 puts in perspective the relative
quantities of radicals formed, directly related to the surge in intensity during a fixed period of
time of 60 minutes (which in all the cases without vacuum corresponds to the peak of
maximum radical concentration obtained at which the inducing light was switched off).
From Table 9 it is easy to see that differences in ESR intensity units, hence in radical
concentration, is much higher for quebracho (111, 101, Table 9) than for all the other three
tannins. The results for the other three tannins are comparable to each other, presenting only
minor differences.
Table 8. Radical decay (=second reaction step) kinetics according to a first order kinetic
law derived from electron spin resonance experiments. (I/Io=a'e-k't).
Table 9. Radical formation reaction. Maximum intensity (10-5) and starting intensity
(10-5) in intensity units of ESR signal and of relative radical concentration
Expressing the same results (Table 9) in percentages show mimosa to give comparable
results to those of quebracho: this gives a faulse idea of the situation because it is the
difference in units which is directly related to the increase in radical concentration on the
tannin, and thus the percentage should not be considered. The percentages are shown in Table
9 to warn about this error in interpretation. These results confirm again that quebracho has the
best antioxidant characteristics, but also show that there is not much difference between the
other tannins.
In the cases with vacuum in which the role of the singlet oxygen is minimized (radical
formation in flavonoids is generally considerably easier in presence rather than absence of air,
hence of singlet oxygen as shown by both more rapid radical formation and decay without
rather than with vacuum in Tables 7, 8 and 9) the real dependence of radical formation from
just the characteristic structure of the tannin can be deduced. Here the difference in units
follows the order
pecan>/=quebracho>>oak>mimosa
It is interesting to relate such a scale to the structural and chemical characteristics of the
three flavonoid tannins in question. Formation of radicals on the flavonoid B-rings generally
leads to heterocycle ring opening, and pyran ring opening is much favourite in
prodelphinidins (pecan) because of their pyrogallol B-ring/phloroglucinol A-ring structure
which ensures better stabilization by delocalisation of the radical on a greater number of
possible sites. For this reason mimosa ( a 70% prorobinetinidin) should then be better than
132 A.Pizzi
quebracho (mainly a profistinidin) which is not the case. There must then be at least another
major structural reaction influencing the above scale. The other frequent bond cleavage
reaction characteristic of flavonoid polymers is the cleavage of the interflavonoid linkage.
Radical formation is also likely to be stabilised through this reaction. Interflavonoid bond
cleavage is relatively easy in pecan and quebracho tannins and notoriously difficult in
mimosa tannin [18,48]. It appears that it is the ease of this reaction superimposed onto the
ease of pyran ring opening which is likely to lead to the scale shown above.
This appears to indicate that in general the higher is the number of -OHs on the flavonoid
B-rings, but particularly the higher is the number of -OHs on the flavonoids A-rings the
greater appears to be the antioxidant behaviour of the flavonoid tannin, although this
characteristic is again overcome by the ease of interflavonoid bond cleavage. The fact that
interflavonoid bond cleavage appear to be strongly related to antioxidant behaviour under
vacuum might not mean that this reaction determines radical formation or stability. It might
only mean that the stereochemistry of tannins which present easier bond cleavage is such, for
instance the structure is more open, that radical formation and uptake are facilitated.
The structural parameters which influence the antioxidant properties of the tannins
change of importance in presence of air, hence in presence of singlet oxygen. The total scale
is mimosa=quebracho>>pecan>/=oak. Here it appears that there is a clear inverse relationship
between the number of A-rings -OHs and of ease of interflavonoid bond cleavage with the
rates of radical formation and decay, namely: in presence of air the greater the number of A-
rings -OHs and the easier the interflavonoid links cleavage the lesser is the antioxidant
activity of the tannin. Thus, the parameters of importance are the same in presence or absence
of air, but the effect is exactly opposite in the two cases. This indicates that another tannin
property might also have a bearing on the antioxidant capability of the tannin, namely its
colloidal state. Flavonoid tannins present decreasing colloidal state according to the scale
mimosa=quebracho>pecan [27]. In presence of singlet oxygen the effect of migration of such
a radical species within the colloidal micelles will afford much more rapid radical formation
or uptake by the tannin, thus improve its radical uptake characteristics (and at the same time
possibly also accelerate radical decay within the micelles). This is indeed the case from the
results in Tables 1 and 2. If the singlet oxygen is not present, hence in absence of air, the
effect of the colloidal state is non-existent for the radical formation reaction and radical
formation is much slower. The effect might have very little bearing on the radical decay
reaction, although with the data available it is impossible to say.
In conclusion the four parameters the combination of which appears to have a bearing on
the antioxidant capabilities of a tannin are (i) the extent of its colloidal state, (ii) the ease of
interflavonoid bond cleavage (or better its stereochemical structure), (iii) the ease of pyran
ring opening and (iv) the relative numbers of A- and B-rings -OH groups. It is the
combination of these four factors which will determine the behaviour as an antioxidant of a
particular tannin under each set of particular application conditions. With the data presently
available it is impossible to quantify the relative extent of the four effects as a function of
application conditions.
Oligomeric Nature, Colloidal State, Rheology… 133
Table 10. First order kinetics of radical decay reaction after radical transfer to tannin
from DPPH, in methanol
The experiments of radical transfer from DPPH to a tannin in solution also gave some
interesting results. Three solvents were tried: tetrahydrofurane was discarded because did not
dissolve the tannins. Dioxane dissolved the tannin but presented problems of radical transfer
between DPPH and tannin: some of the few reliable results in dioxane are reported in Table
10. Solutions of tannin and DPPH in methanol instead gave reliable results: these are also
shown in Table 10. These show that as regards the radical decay reaction mimosa is slower,
thus has better antioxidant power than quebracho, this result closely matching and supporting
what already obtained by radical transfer in solvent with the stopped-flow apparatus
experiments [44]. This result supports again the use of ESR techniques for this type of
determination. As regards the other tannins the increasing order of the rate of the radical
decay reaction (thus passing from the slowest to the fastest radical decay rate) (Table 10) is as
follows
gambier<pine=/<mimosa=/<oak<pecan<quebracho
which presents quite a different order from the experiments done without solvent and simply
by light irradiation. The order of quebracho, pecan and oak in the above scale reproduces
what obtained without vacuum in the experiments without solvent, except for the relative
position of mimosa tannin in the scale which is now completely different. It is clear then, that
solvation parameters also appear to play an important role under certain conditions. This goes
to shaw the complexity of the interrelation of parameters in tannins radical reactions. As
regards the radical formation reaction the results obtained can be calculated only in a very
few cases, the reaction in the other cases being either too fast or too unreliable (Table 10).
membranes, inhibiting any activity they might have. Thus, pharmaceutical containing tannins
and aimed at curing bacterial intestinal infections have been around already for some time.
Some studies on their anticaries effectiveness have also been conducted [49]. Independently
from these, almost obvious, pharmaceutical applications of tannins several experimental
studies on their use for other pharmaceutical/medical applications have been reported.
Particularly well reported are the studies on their antitumor and anticancer activity [50-53].
More recently, work on their antiviral effectiveness has been investigated [54,55]. The data
which follow in Tables 11-18 are the preliminary results obtained on the the antiviral activity
of 12 different flavonoid and hydrolysable tannins which were carried out by the medical
dept. of Leuven university [54,55].
Table 11. Tannins concentration required to protect CEM cells against the
cytopathogenicity of HIV by 50 %
EC50 = effective concentration or concentration required to protect CEM cells against the
cytopathogenicity of HIV by 50 %
Oligomeric Nature, Colloidal State, Rheology… 135
Table 12. Cytotoxicity and antiviral activity of compounds in HEL cell cultures,
Herpes and vesicular stomatitis viruses. Tannins added before virus administration
The results in Tables 11-18 evaluate both the effectiveness of 12 different tannins as
measured by the Minimum Inhibitory Concentration (MIC) of the tannin required to reduce
virus-induced cytopathogenicity by 50 %. The lower is the MIC value the better is the
compound as an antiviral substance.
Equally important, the results in the tables measure the Minimum Cytotoxic
Concentration (MCC) required to cause a microscopically detectable alteration of normal cell
morphology. The higher the MCC the less toxic to the patient’s cells is the compound and the
better is the compound as an antiviral substance.
136 A.Pizzi
Thus, what is looked for is the lowest possible MIC and the highest possible MCC. These
results are in vitro screening tests. A good results must be still translated into being effective
by the carrier used to deliver it and the way the substance is delivered to the relevant site
where it is needed. Nonetheless as these are very advanced results and they should be
recorded , they are reported in full here. It is evident that different tannins can be very
effective against different viruses, polyphenolic grouping being the cause of this behaviour.
Most likely, they tan the proteins and they associate with the carbohydrates of the virus
membrane. In all an effect similar to their well known association with hide proteins to give
leather and with carbohydrates.
Oligomeric Nature, Colloidal State, Rheology… 137
Table 14. Inhibitory effects of tannins on the proliferation of murine leukemia cells
(L1210/0), murine mammary carcinoma cells (FM3A) and human T-lymphocyte cells
(Molt4/C8, CEM/0)
Table 15. Cytotoxicity and antiviral activity of compounds in HEL cell cultures,
influenza viruses
Table 16. Cytotoxicity and antiviral activity of compounds in HEL cell cultures,
Corona viruses
Table 17. Cytotoxicity and antiviral activity of compounds in HEL cell cultures,
Herpes and Vaccinia viruses. Tannins added after virus administration
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Editor: Raymond B. Keller © 2009 Nova Science Publishers, Inc.
Chapter 4
GRAPEFRUIT FLAVONOIDS:
NARINGIN AND NARINGININ
ABSTRACT
Naringin is the flavonoid compound found in grapefruit that gives grapefruit its
characteristic bitter flavor. Grapefruit processors attempt to select fruits with a low
naringin content, and often blend juices obtained from different grapefruit varieties to
obtain the desired degree of bitterness. Naringin is believed to enhance our perception of
taste by stimulating the taste buds; some people consume a small amount of grapefruit
juice before a meal for this reason.
Naringin and its aglycone naringinin are commonly used health supplements; they
exert a variety of biological actions. This article attempts to review their
pharmacokinetics and pharmacological actions from scientific publications up to
November 2008 including effects on the cardiovascular system, on the skeletal system,
on smooth muscle, on the gastric intestinal system, on the endocrine system, also effects
against tumours, protection against toxins in chemotherapy drugs and the environment,
antioxidant effects, drug interactions, anti-inflammatory effects, and the newly
discovered osteogenic and antibacterial actions.
INTRODUCTION
Naringin is the flavonoid compound found in grapefruit that gives grapefruit its
characteristic bitter flavor. Grapefruit processors attempt to select fruits with a low naringin
142 Ricky W. K. Wong and A. Bakr M. Rabie
content, and often blend juices obtained from different grapefruit varieties to obtain the
desired degree of bitterness. Naringin is believed to enhance our perception of taste by
stimulating the taste buds (some people consume a small amount of grapefruit juice before a
meal for this reason). Naringin may be instrumental in inhibiting cancer-causing compounds
and thus may have potential chemotherapeutic value. Studies have also shown that naringin
interferes with enzymatic activity in the intestines and, thus, with the breakdown of certain
drugs, resulting in higher blood levels of the drug. A number of drugs that are known to be
affected by the naringin in grapefruit include calcium channel blockers, estrogen, sedatives,
medications for high blood pressure, allergies, AIDS, and cholesterol-lowering drugs.
Caffeine levels and effects of caffeine may also be extended by consuming grapefruit or
grapefruit juice. While the effect of naringin on the metabolism of a drug can increase the
drug's effectiveness, it can also result in dosages that are inadvertently too high. Therefore, it's
best not to take any drugs with grapefruit juice unless the interaction with the drug is known.
In addition, the effects of drinking grapefruit juice is cumulative, which means that if you
drank a glass of grapefruit juice daily with your medication for a week, the drug interaction
would be stronger at the end of the week than at the beginning.
Research on naringin and naringinin shows the following effects:
∗
Tel: 852-28590554; Fax: 852-25593803; E-mail: fyoung@hkucc.hku.hk
Grapefruit Flavonoids: Naringin and Naringinin 143
Reference
Study The potency, structure-activity relationship, and mechanism of
vasorelaxation of flavonols: fisetin, rutin, quercetin; flavones: chrysin,
flavone, baicalein; flavanones: naringenin, naringin; isoflavones: diadzein
and flavanes: epigallo catechin gallate, were examined in the isolated rat
aorta.
Results Most of the flavonoids tested showed concentration dependent relaxant
effects against K+ (80 mM) and phenylephrine (PE, 0.1 microM)-induced
contractions with a greater inhibition of the responses to the alpha1-
adrenoceptor agonist.
Conclusion Relaxant effects of flavonoids on vascular smooth muscle of the isolated
rat thoracic aorta.
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Editor: Raymond B. Keller © 2009 Nova Science Publishers, Inc.
Chapter 5
ABSTRACT
Numerous epidemiological studies show that some nutrients may protect against
vascular diseases, cancers and associated inflammatory effects. Consequently, the use of
phytoconstituents, namely those from the human diet, as therapeutic drugs is relevant.
Various studies report the efficiency of phyto-molecules which have cellular targets
similar to those of the new drugs developed by pharmaceutical companies. Indeed, more
than 1600 patents are currently reported concerning flavonoids and 3000 patents
concerning polyphenols. Pleiotropic pharmaceutical activities are claimed in fields such
as cancer, inflammation arthritis, eye diseases and many other domains. The increase of
activities after combination with other natural compounds or therapeutic drugs is also
patented. In addition, the aforementioned molecules, from natural origin, generally
exhibit low toxicities and often a multipotency which allow them to be able to
simultaneously interfere with several signalling pathways. However, several in vivo
studies revealed that polyphenols / flavonoids are efficiently absorbed by the organism,
but unfortunately have a low level of bioavailability, glucuronidation and sulphation
being limiting factors. Therefore, many laboratories are developing elements to increase
bioavailability and consequently the biological effects of these natural molecules. For
example, the modifications in the lipophilicity of molecules increase the cellular uptake
and consequently involve a best absorption without loss of their activities. Moreover
∗
Phone : 33 3 80 39 62 37, Fax : 33 3 80 39 62 50, Email : ddelmas@u-bourgogne.fr
182 Dominique Delmas, Frédéric Mazué, Didier Colin et al.
A) INTRODUCTION
A wide variety of plant-derived compounds, including polyphenols and flavonoids, is
present in the human diet and may protect against vascular diseases, cancers and associated
inflammatory effects (Figure 1).
The impetus sparking of this scientific inquiry was the result of many epidemiologic
studies. For example, in France, as compared with other western countries with a fat-
containing diet, the strikingly low incidences of coronary heart diseases is partly attributed to
the consumption of red wine, which contains high levels of polyphenols [1]. Similarly,
benefit effects may be attributed to the flavonoids of green tea.
Figure 2. Polyphenols effects on tumor initiation. Polyphenols are able to prevent initiation phase by
inhibition of carcinogen activation (R+) induction of carcinogen deactivation and subsequently
blocking interaction between DNA and carcinogen (R+).
For example, resveratrol and genistein are able to antagonize the transactivation of genes
regulated by the aryl hydrocarbon receptor (AhR) ligand, such as the 7,12-
dimethylbenz[a]anthracen (DMBA) [6]. Consequently, compounds are able to reduce the
number of DNA adducts induced by various chemical agents.
Moreover, polyphenolic compounds and flavonoids could induce phase II enzyme which
generally protects tissues and cells from endogen and/or exogen intermediate carcinogens.
Activation of phase II detoxifying enzymes by flavonoids / non flavonoids, such as UDP-
glucuronyl transferase [7-9], glutathione S-transferase (GST) [10], quinone reductase [11,
12], and sulfotransferase [13] result in the detoxification of carcinogens and represent one
mechanism of their anticarcinogenic effects (Figure 2).
For example, Kong et al. propose a model where an antioxidant such as butylated
hydroxyanisol (BHA) and isothiocyanate sulforafane (SUL) may modulate the mitogen-
activated protein kinases (MAPKs) pathway leading to transcriptional activation of the
nuclear factor erythroid 2p45 related factor, Nrf2 (a basic leucine zipper transcription factor)
and of the antioxidant electrophile response element (ARE), with subsequent induction of
phase II detoxifying enzymes such as hemeoxygenase (HO-1), GST, NAD(P)H:quinine
reductase (NQO-1) [14].
Recently, quercetin was reported to protect human hepatocytes from ethanol-induced
cellular damage and this beneficial effect was mediated by a pathway involving extracellular
signal-regulated protein kinase (ERK), p38, and Nrf2, with the induction of HO-1 expression
[15].
In addition, the activation of Nrf2 up-regulated the induction of a phase II enzyme, NQO-
1, in HepG2 cells [16]. In the same manner, resveratrol is able to up-regulate NQO-1 gene
expression in human ovarian cancer PA-1 cells [17] by its activation of kinase pathways.
Development of Promising Naturally Derived Molecules… 185
Furthermore, a study using a resveratrol affinity column shows that the dihydronicotinamide
riboside quinone reductase 2 (NQO-2) binds resveratrol and could constitute a potential target
in cancer cells [18].
The promotion phase is the second major step in the carcinogenesis process in which
specific agents (referred to as promoters) trigger the further development of the initiated
cells. Promoters often, but not always, interact with the cellular DNA and influence the
further expression of the mutated DNA so that the initiated cell proliferates and progresses
further through the carcinogenesis process (Figure 3).
An intricate network of signalling pathways is involved in these control mechanisms,
especially the cell cycle and the induction of apoptosis. This induction of cell death in
precancerous or malignant cells is considered to be a promising strategy for chemopreventive
or chemotherapeutic purpose. Natural compounds, like many cytotoxic agents, affect cell
proliferation by disturbing the normal progress of the cell cycle. In fact, both stilbenes and
flavonoids are able to block cell progression through the cell cycle, this blockage depends on
the cell type, the natural compound concentration, and the treatment duration. Various studies
report that checkpoint at both G1/S and G2/M of the cell cycle is found to be perturbed by the
phyto chemicals [19-21]. Checkpoints are controlled by a family of protein kinase complexes,
and each complex is composed minimally of a catalytic subunit, cyclin-dependent kinases
(cdks), and its essential activating partner, cyclin.
Figure 3. Polyphenols effects on tumor progression. Polyphenols are able to prevent promotion phase
by inhibition of cell cycle progression and are able to induce apoptosis.
186 Dominique Delmas, Frédéric Mazué, Didier Colin et al.
Cyclins play a key regulatory role in this process by activating their partner cdks and
targeting them to the respective protein substrates [22]. Complexes-formed in this way are
activated at specific intervals during the cell cycle and their inhibition blocks the cell cycle at
the corresponding control point. These key regulators can be affected by these flavonoids and
stilbenes leading to an arrest of the cell cycle. For example, epigallocatechin-3-gallate
(EGCG), a green tea polyphenol, mediates a G1 phase arrest in various cancer cell lines such
as ovarian, pancreatic through a modulation of cyclin D1 and p21WAF1 [23, 24]. The balance
between pro- and anti-apoptotic Bcl-2 family proteins favors apoptosis in prostate cancer cells
also arrested in G0/G1 phase [25]. In the same manner, treatment by curcumin, resveratrol or
silymarin involve an increase of p21WAF1 and p27KIP1 expressions and inhibit the expression
of cyclin E and cyclin D1, and hyperphosphorylation of retinoblastoma (Rb) protein [26-31].
Concerning the G2/M phase, some phytochemicals can arrest the cell cycle at the
transition from G2 stage to M stage such as genistein [32-35], resveratrol [36-38], curcumin
and quercetin [39] in various cancer cell types (Figure 3). Biochemical analysis demonstrates
that the disruption of G2 phase progression by polyphenolic compounds is accompanied by
an upregulation of p21WAF1 and an inactivation of cyclin-dependent kinase, Cdk1 [31, 35, 36,
38, 40].
In addition to cell cycle arrest, another specialized event of polyphenol compounds action
involves the induction of apoptosis (Figure 3). Induction of apoptosis in precancerous or
malignant cells is considered to be a promising strategy for chemopreventive or
chemotherapeutic purposes. The induction of apoptosis triggered by polyphenolic compounds
has been observed in various cell types with different pathways. Indeed it has been
demonstrated that polyphenols are able to activate cell death by the mitochondrial pathway or
by the death receptor pathway.
The mitochondrial pathway is activated in response to extracellular signals and internal
disturbances such as DNA damages. We and others have shown that polyphenols such as
resveratrol [41], quercetin [42, 43], genistein [44-47] induce apoptosis in various tumor cell
lines by modulating pro-apoptotic Bcl-2 family proteins which are known as "BH3-only
proteins" behaving as sensors of cellular damage and initiating the agents of death process.
We and others have shown that polyphenols and flavonoids down-regulate Bcl-2 protein
expression [41, 48-53] and gene expression [54-56], which normally stabilizes the
mitochondrial potential of the membrane (Δϕm), and inhibits ROS production. Cytochrome c,
in the cytosol, induces oligomerization of the adapter molecule Apaf-1 to generate a complex,
the apoptosome, in which caspase-9 is activated. Active caspase-9 then triggers the catalytic
maturation of caspase-3 and other resultant caspases, thus leading to cell death.
Major external signals triggering apoptosis are mediated by receptor/ligand interactions
(such as CD95 and tumor necrosis factor receptor). The binding of ligand to receptor induces
receptor clustering and the formation of death inducing signalling complex (DISC). This
complex recruits via the adaptator FADD (Fas-associated death domain protein), multiple
procaspase-8 molecules resulting in caspase-8 activation. It can activate the proteolytic
cascade or / and converge on the mitochondrial pathway through the activation of pro-
apoptotic members of the Bcl-2 family. We and others have shown that
polyphenols/flavonoids can involve this pathway in various cells lines.
The action of different polyphenols/flavonoids on the cell cycle at different stages, on the
apoptosis cascade, or on the metabolizing enzymes, depends on their cellular targets, the
concentrations used and the cellular type. These findings suggest a very specific mechanism
Development of Promising Naturally Derived Molecules… 187
Finally, stilbenes and flavonoids can act on the third step of carcinogenesis, the
progression which is associated with the evolution of the initiated cells into a biologically
malignant cell population. In this stage, a portion of the benign tumor cells may be converted
into malignant forms leading to a true cancer. At this stage, tumor progression is certainly too
advanced for chemopreventive intervention but not for a chemotherapeutic intervention.
During tumor progression, polyphenols (resveratrol, curcumin, EGCG, silymarin, …), as
previously described, can act as antiproliferative agents by blocking cell cycle progression
and inducing apoptosis of cancer cells. The phytochemical compounds can also act on events
more specific of the progression / invasion step. In this final stage, invasion, can break away
and start new clones of growth distant from the original site of development of the tumor. It is
reported that resveratrol, quercetin, genistein show an anti-proliferative effect on highly
invasive breast carcinoma cells (MDA-MB-435) [45, 49, 57]. In fact, these polyphenols can
modify the key regulators of angiogenesis such as vascular endothelial growth factor (VEGF)
and could inhibit matrix metalloproteinase. Indeed, resveratrol, genistein, quercetin and
silymarin inhibit VEGF expression in various cancer cell lines [58-67]. One hypothesis is that
polyphenols inhibition of VEGF-induced angiogenesis is mediated by disruption of ROS-
dependent Src kinase activation and the subsequent VE-cadherin tyrosine phosphorylation.
Efficient tumor invasion also requires partial degradation of the extracellular matrix (ECM) at
the invasion front. The matrix metalloproteinases (MMPs) are the main proteases involved in
remodeling the ECM contributing to invasion and metastasis, as well as tumor angiogenesis
[68]. Concerning the human MMPs, the expression levels of gelatinase-A (MMP-2) and
gelatinase-B (MMP-9) are associated with tumor metastasis for various human cancers [69,
70]. Resveratrol [71-73], silibin [74] and genistein [75, 76] are able to decrease the MMP-9
expression via diminished MAP kinases activation.
reach higher values for catechins from green tea, isoflavones from soy, flavanones from citrus
fruits or anthocyanidins from red wine (3–26%). Interindividual variations have also been
observed: 5–57% of the naringin consumed with grapefruit juice is found in urine according
to the individual [81]. A major part of the polyphenols ingested (75–99%) is not found in
urine. This implies they are not absorbed through the gut barrier, but excreted in the bile or
metabolized by the colonic microflora or other tissues. In the same manner to chemical drugs,
animal cells react to plant polyphenols exposure by recognizing these molecules as
xenobiotics. As consequence, the cells transform these compounds in order to eliminate them
as quick and as extended as possible. The pharmacokinetic is an essential parameter to select
natural compounds based on their biological activity, especially their possible anticancer
properties. For example, resveratrol is a well known promising anticancer natural molecule
[82], but this molecule exhibits a low bioavailability [9, 83]. To overcome this problem, it is
interesting to used: 1) phytochemicals combinations, 2) phytochemicals / therapeutics drugs
association, 3) chemically modified natural polyphenols.
1. Phytochemical Combinations
The synergistic activity of phytochemical combinations can be easily tested in vitro and
the combination treatment might represent a new strategy that can play a major role in the
future of cancer chemoprevention [84]. Various reports show that the combination of several
natural molecules or one phytochemical compound with an anticancer drug might be more
effective in cancer prevention or in cancer therapy than a single molecule (Table I). By their
pleiotropic actions, the use of several polyphenols can operate many cellular targets and/or
decrease the phenomena of metabolisation, glucuronidation and sulfation, being limiting
factors, and consequently increase the bioavailability and the biological effects of these
natural molecules. It is the case for the EGCG glucuronidation [85] which is inhibited by the
association with an alkaloid derived from black pepper, the piperine in the small intestine
[86]. So, the bioavailability of EGCG is enhanced in mice. In a same manner, it is reported
that coadministration of piperine and curcumin to humans and rats enhanced the
bioavailability of curcumin by 2000% and 154%, respectively [87]. The use of flavonoids can
also enhance the bioavailability of other polyphenols such as resveratrol. Indeed, many
flavonoids can inhibit the hepatic glucuronidation and the hepatic/ duodenal sulphation of
resveratrol and such inhibition may improve the bioavailability of this compound [88-90].
The modifications of bioavailability can enhance the toxicity of phytochemicals on
cancer cells through synergistic or additive actions. For example, an antiproliferative synergy
between resveratrol, quercetin and catechin is observed on human breast cancer cells [91]. No
modifications is observed when compounds are used alone, but at only 0.5 µM, they
significantly reduce cell proliferation and block cell cycle progression in vitro and at 5 mg/kg
reduce breast tumor growth in a nude mouse model. The essential steps in tumor progression
are the cell cycle and apoptosis alteration (see previously). Ellagic acid and quercetin interact
synergistically with resveratrol in the induction of apoptosis and cause transient cell cycle
arrest in human leukemia cells [92]. This apoptosis effect may be due to p21 overexpression
and a greater p53 phosphorylation [93]. The MAP kinases, JNK1,2, and p38 are also activated
in a “more than additive” manner.
Development of Promising Naturally Derived Molecules… 189
quercetin 5 µM / 10 µM Antiproliferation,
synergy [97]
ellagic acid each Cytotoxicity, Apoptosis
MDA-MB- resveratrol
231 (human quercetin Antiproliferation,
0,5 µM each synergy [91]
breast cancer Apoptosis
cells) catechin
The caspase-3 activity can be synergistically induced by combinations of ellagic acid and
reveratrol (combination index 0,64) or quercetin and resveratrol (combination index 0,68)
[92]. A combination of EGCG and curcumin synergistically suppresses MDA-MB-231
estrogen receptor-alpha-breast cancer cells by cytotoxic effects and cell cycle arrest [94].
All those studies lead to think that polyphenols are mostly efficient against cancer when
used in combinations or with other natural compounds in a plant-rich diet [95-98].
For example, the green tea polyphenol epigallocatechin-3-gallate shows synergy with 5-
fluorouracil on human carcinoma cell lines [99]. Besides EGCG, various flavonoids or
stilbenes can present a synergy or an additive action with anticancer drugs through a
modification of anticancer drug metabolism or multiple actions on various targets. For
example, various phenolic antioxidants (catechin, epicatechin, fisetin, gallic acid, morin,
myricetin, naringenin, quercetin and resveratrol) can modify the paclitaxel metabolism.
Indeed, Paclitaxel metabolites are virtually inactive in comparison with the parent drug. Some
reports show that phenolic substances might increase paclitaxel blood concentrations during
chemotherapy by inhibiting. cytochrome p450-catalyzed metabolism of paclitaxel [100].
Moreover the enhancement of paclitaxel action can be due to a complementary effect on
targets [101, 102]. In resistant non-Hodgkin's lymphoma (NHL) and multiple myeloma
(MM), resveratrol and paclitaxel selectively modify the expression of regulatory proteins in
the apoptotic signalling pathway. Indeed, combination treatment results in apoptosis through
the formation of tBid, mitochondrial membrane depolarization, cytosolic release of
cytochrome c and Smac/DIABLO, activation of the caspase cascade, and cleavage of
poly(adenosine diphosphate-ribose) polymerase. Combination of resveratrol with paclitaxel
have minimal cytotoxicity against quiescent and mitogenically stimulated human peripheral
blood mononuclear cells. Inhibition of Bcl-x(L) expression by resveratrol is critical for
chemosensitization and its functional impairment mimics resveratrol-mediated sensitization to
paclitaxel-induced apoptosis. Interestingly, a simultaneous exposure does not amplify the
antiproliferative or pro-apoptotic effects of paclitaxel [103]. In fact, a pretreatment with
resveratrol induces p21WAF1 expression suggesting a possible arrest of cell cycle favoring the
effect of paclitaxel action. This could be the case of the combination with 5-fluorouracil (5-
Fu) which is a classic drug used in colorectal and hepatoma chemotherapy. Indeed, it was
reported that resveratrol can exert synergic effect with this drug to inhibit hepatocarcinoma
cell proliferation by the induction of apoptosis [104, 105].
Concerning cytokines, we and others have shown that polyphenols (resveratrol,
quercetin, kaempferol, silibinin, genistein, apigenin) are able to sensitive to TRAIL (tumor
necrosis factor-related apoptosis-inducing ligand)-induced apoptosis in cancer cells [106-
114]. For example, in human colon cancer cell lines, some phytochemicals, resveratrol,
quercetin, kaempferol can sensitize these cells to TRAIL-induced apoptosis [106, 108, 109,
112]. This sensitization by quercetin and resveratrol involves an increase of formation of a
functional DISC at plasma membrane level [108, 112] and activates a caspase-dependent
pathway that escapes Bcl-2-mediated expression [112]. The cholesterol sequestering agent
nystatin prevents resveratrol or quercetin-induced death receptor redistribution and cell
sensitization to death receptor stimulation, suggesting that polyphenols-induced redistribution
of death receptors in lipid rafts is an essential step in their sensitizing effects expression [108,
112]. The sensitization by resveratrol involves also a cell cycle arrest-mediated survivin
depletion and an upregulation of p21 [106].
Previous studies have documented that stilbenes and flavonoids, despite an efficient
absorption by the organism, have unfortunately a low level bioavailability, glucuronidation
and sulfation being limiting factors [78-80].
Development of Promising Naturally Derived Molecules… 191
Figure 4. Chemical structures of resveratrol and derivatives. Chemical structures of Trans (A) or Cis
(B) resveratrol (R=OH); resveratrol triacetate (R=CH3COO); trimethoxyresveratrol (R=CH3O).
conversion and have a higher bioavailability with respect to resveratrol. Recently, Saiko et al.,
reported the influence of several trans-resveratrol analogs on HT 29 human colon cancer cell
proliferation inhibition and apoptosis [123]. Indeed, a poor effect with 3,5, 4’,5’
tetramethoxy-trans-resveratrol is observed while a strong effect of 3,5 4’ trimethoxy-trans-
resveratrol and of 3,3',4,5'-tetramethoxy-trans-stilbene lead to remarkable changes of the cell
cycle distribution on HT29 cells. Interestingly, after treatment with 3,5 4’ trimethoxy-trans-
resveratrol, growth arrest occurrs mainly in the G2-M phase, whereas incubation with
3,3',4,5'-tetramethoxy-trans-stilbene resulted in arrest in the G0-G1 phase of the cell cycle.
The presence of hydroxylated group does not significantly change the antiproliferative effect.
This is in agreement with results of Ovesna et al., reporting that hydroxylation mainly protect
against DNA damage [124]. However experiments are done on established cell lines which
are already initiated. The only visible effect can be seen on promotion step.
Moreover, the isomerisation could modulate the activity of these compounds. Indeed,
methoxylated Z-stilbenoids have a structural analogy with combretastatin A4, a potent
antimitotic which interacts with tubulin like colchicines [125]. In the majority of cases where
pairs of E- and Z-isomers were evaluated for antitumor activity, the cis (Z)-isomers proved
significantly more active effects than their trans (E) analogs; nevertheless, the
antiproliferative / apoptotic activity ratio between the E/Z isomers reported show wide
variations and in some cases both either have comparable activities or the E-isomer may be
even more active, as for E and Z-resveratrol. The strong effect of cis (Z) 3,5,4’-
trimethylresveratrol is due to its inhibition of the microtubule polymerisation [126, 127],
leading to a blockade of the cell division. This blockade provokes an increase of cell death,
probably by mitotic catastrophe [128]. Other compounds exhibit a similar antiproliferative
activity to trans (E) resveratrol, but their action mechanism is very different: resveratrol
accumulates cells in S phase, while most of the other synthetic derivatives stop mitosis (M
phase). The stronger effect of cis (Z) methoxy derivatives than the trans (E) counterparts is
not linked to the lack of anti-oxidative effect (diseappearence of hydroxyl groups) but would
be due to a steric mechanism leading to interference with different pathways as compared to
the trans derivatives.
When the analogs of phenolics compounds is used, it is important to choose the methods
to estimate the biological effects such as antiproliferative activity. Indeed, we and others have
shown that methods commonly used to measure cytotoxic and/or antiproliferative effects
could lead to a pitfall resulting from a differential sensitivity to natural compounds [117,
129]. For example, in the human colorectal cancer cell, SW480, we observed that resveratrol
triacetate and a preparation containing resveratrol (16 %) and ε-viniferin (20 %) can induce
an increase in the MTT-reducing activity [117]. These observations are very important since
the MTT test can mask antiproliferative activities and could be a possible pitfall in cell
sensitization determination. In genistein-treated cells, it was suggest that this increase of
MTT-reducing activity could be due to an increase of the cell volume and of the number of
mitochondria [130]. Similarly, resveratrol and its acetylated form induce an increase in cell
volume and also induce an accumulation of SW480 cells in S phase [117]. Bernhard et al.
[129] suggest that this phenomenon is associated with the differentiation process. This
hypothesis is supported by the ability of resveratrol to induce differentiation of colon
carcinoma cells via nuclear receptor [131]. However, it was not the case with ε-viniferin and
its acetylated form which have no effect on the cell volume and differentiation, probably
Development of Promising Naturally Derived Molecules… 193
because these polyphenols may interact with the redox activities of mitochondria and
consequently contribute to the reduction of MTT.
Consequently, it is necessary to evaluate methods used for cytotoxic determinations and
to evaluate the absence of toxicities in normal cells. Several studies have shown that
polyphenols compounds such as resveratrol and viniferin have no cytotoxic effect on normal
hematopoietic progenitor cells, in contrast to leukemic cells. In addition, resveratrol shows
specific cytotoxic effects toward tumor cells when compared with normal lung [132] and
blood cells [133]. Furthermore, at the concentrations inducing growth inhibition and cell
cycle arrest in colon carcinoma cells, polyphenols compounds and vineatrol did not impair the
viability of human normal peripheral blood mononuclear cells (PBMC) and much higher
concentrations were required to induce a cytotoxic effect in these cells [52], but what are the
effects of derivatives compounds on the normal cells ? This point is very critical for a
potential use of these compounds in therapeutics and more studies should be carried out to
determine the absence of toxicity on normal cell and in animal models.
1. Production Methods
New plants are reported to be powerful producers of polyphenols with strong activity in
traditional pharmaceutical domains (inflammation, cancer, cardiovascular…) including algae
(EP1977756) and a new plant species from Fallopia named igniscum (DE102007011676)
(Table II).
2. Modification of Structures
Syntheses of new analogs of polyphenol remain a good approach for innovation (Table
IV). In CA2617213 inhibition of inflammation, recruitment and cell proliferation is claimed
according to chemical modifications. Rather complex compounds are also reported (example
in EP1901735 or CN101115763) with questionable possibility for final pharmaceutical
production (Figure 6A). Chemical modification of resveratrol remains hardly explored
especially in anticancer and cardiovascular fields (see US2005240062 as an example).
Association of polyphenols with fatty acids is again reported for increased activity
(US2008176956 and WO2007099162 ).
A)
EP1901735
CN101115763 WO2007099162
B)
3. Formulation
Polyphenol administration is complex due to high level of astringency and bitterness and
low absorption associated with questionable gut stability. Therefore new ways for increasing
compliance and oral availability are under study in many companies. Microencapsulation
(US2008213441) or association with new vehicle (WO2008072155) as well as
nanoencapsulation (CN101214225) and nanocrystallization (CN101195559) are good ways to
increase stability and facilitate absorption (Table VI).
If required optimization of topical use of polyphenol is also hardly worked for the
treatment of skin disorders. S2008095866 claims increase of topical availability of
polyphenols with phosphorylated polyphenols. An original approach with the synthesis of
bioprecursors of polyphenol is also reported in EP1893555 especially for topic targets. In that
case bioprocessing of these molecules will lead to in situ production of active polyphenols.
Food industry is also concerned by polyphenol use as demonstrated by US2008213456
showing incorporation of extracts into bars (Table VI).
Flavonoid usage is also complex due to instability of the compounds. Topical use is
limited due to interaction with UV light, therefore stabilization of the drug with antioxidant
compounds is necessary (WO2008140440) (Table VII).
In order to increase oral availability mixture with pectin from citrus and other origin is
also reported (JP2008174553). Modification of taste and increase of water solubility is
200 Dominique Delmas, Frédéric Mazué, Didier Colin et al.
4. Combination Treatment
Figure 7. Synergy of activity after mixing polyphenol and conjugated fatty acids on prostate cancer
cells. (US2008234361) (according to patent number US2008234361).
Development of Promising Naturally Derived Molecules… 201
The patent approach remains especially active in the polyphenol field. New formulations
and mixtures seem to be especially appreciated. New mechanisms of action in relation with
202 Dominique Delmas, Frédéric Mazué, Didier Colin et al.
published scientific results can be observed (sirtuin phenomenon). The number of patents is
relatively stable according to a year of filing, indicating that the situation will remain
relatively stable for the future.
CONCLUSION
We have established that one of the goals is to increase bioavailability and consequently
the biological effects of natural molecules. Increase in the lipophilicity of molecules should
improve the cellular uptake and consequently lead to better absorption without loss of their
activities. We presented the idea that isomerisation and methylation of hydroxyl groups of
some polyphenols especially resveratrol are crucial to improve the molecule efficiency in
blocking cell proliferation by changing the cell molecular targets. We focused on the
relevance of using flavonoid and polyphenol combinations or chemical modifications to
enhance their biological effects. We suggested innovative directions to develop new types of
drugs which may especially be used in combination with other natural components or
pharmacological conventional drugs in order to obtain a synergistic effect.
ACKNOWLEDGMENTS
This project was supported by the "Conseil Régional de Bourgogne", BIVB and Ligue
contre le Cancer, comités Côte d’Or et Jura.
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In: Flavonoids: Biosynthesis, Biological Effects… ISBN: 978-1-60741-622-7
Editor: Raymond B. Keller © 2009 Nova Science Publishers, Inc.
Chapter 6
ABSTRACT
Cocoa has recently become an object of interest due to its high content of flavonoids,
mainly the monomers epicatechin and catechin and various polymers derived from these
monomers called procyanidins. Previous in vitro studies have shown the ability of cocoa
flavonoids to down-regulate inflammatory mediators produced by stimulated
macrophages, but there are no studies that consider the effects of in vivo cocoa intake on
inflammatory response. In the present article, we report the in vivo cocoa inhibitory effect
on the acute inflammatory response. Female Wistar rats received Natural Forastero cocoa
containing 21.2 mg flavonoids/g for 7 days (2.4 or 4.8 g per rat kg, p.o.). Then, acute
inflammation was induced by means of carrageenin, histamine, serotonin, bradykinin or
PGE2 hind-paw injection. Rats fed 4.8 g/kg/day cocoa showed a significant reduction in
the hind-paw edema induced by carrageenin from the first hour after induction (P<0.05).
However, cocoa intake did not modify the edema induced by histamine, serotonin or
PGE2. Only a certain protective effect was observed at the lowest dose of cocoa in the
bradykinin model. Moreover, peritoneal macrophages from rats that received
4.8 g/kg/day cocoa for 7 days showed a reduced ability to produce radical oxygen species
(ROS), nitric oxide (NO), tumor necrosis factor α (TNFα) and interleukin 6 (IL-6). This
fact could justify, at least partially, the beneficial effect of cocoa on carrageenin-induced
inflammation. In summary, a diet rich in cocoa flavonoids was able to down-regulate the
acute inflammatory response by decreasing the inflammatory potential of macrophages.
∗
E-mail: margaridacastell@ub.edu
214 M. Castell, A. Franch, S. Ramos-Romero et al.
1. INTRODUCTION
One of the foods with a relatively high content of flavonoids is cocoa, which is obtained
from the beans of the Theobroma cacao tree (Lee, 2003). The beneficial effects of cocoa were
known as early as 600 BC: the Mayans and Aztecs roasted and ground cocoa beans to prepare
a divine beverage called xocolatl, which was mainly used to cure fatigue, fever, infections,
and heart pain (Hurst et al., 2002). Although most people presently see cocoa and its
derivatives only as snacks, scientific evidence of the health benefits of cocoa known by the
ancients is emerging now.
In addition to being a rich source of fiber (26–40%), proteins (15–20%), carbohydrates
(∼15%), and lipids (10–24%), cocoa powder contains minerals, vitamins (A, E, B and folic
acid) and a high amount of flavonoids. However, cocoa flavonoid content is difficult to
establish because it depends on geographic origin, climate, storage methods and
manufacturing processes (Manach et al., 2004; McShea et al., 2008). Cocoa powder mainly
contains the flavanols (-)-epicatechin, (+)-catechin and polymers derived from these
monomers called procyanidins; it is reported to contain up to 70 mg/g of polyphenols (Vinson
et al., 1999). Epicatechin and catechin are biologically active, but epicatechin is more
efficiently absorbed than catechin (Baba et al., 2001). Procyanidins are the major flavonoids
in cocoa and chocolate products ranging from 2.16 to 48.70 mg/g (Gu et al., 2006). Short
procyanidins (dimers and trimers) are absorbed in the small intestine and rapidly detected in
plasma (Baba et al., 2000). However, large procyanidins are less efficiently absorbed in its
polymeric form, but can be metabolized by colon microflora to phenolic acids and then
absorbed (Manach et al., 2004). Quercetin and its derivatives, naringenin, luteolin and
apigenin, are also present in smaller quantities (Sanchez-Rabaneda et al., 2003).
Experimental and clinical data suggest that the consumption of cocoa flavonoids can
produce positive clinical benefits in the cardiovascular system (review by Buijsse et al., 2006
and Cooper et al., 2008) and also in brain function (reviewed by McShea et al., 2008). Cocoa
intake reduces the risk of cardiovascular disease by modulating blood pressure (Grassi et al.,
2005; Taubert et al., 2007) as well as decreasing blood cholesterol (Baba et al., 2007),
moreover it produces vasodilatation and inhibits platelet activation and aggregation (Hermann
et al., 2006). In vitro assays and studies in animal models suggest that cocoa has beneficial
effects on neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease
(Datla et al., 2007; Ramiro-Puig et al., 2009a). However, it remains to establish doses and
length of treatment because a recent trial in healthy adults does not find neuropsychological
effects after a short-term dark chocolate intake (Crews et al., 2008). Biological effects of
cocoa are mainly attributed to the high content of antioxidant polyphenols (reviewed in
Ramiro-Puig et al., 2009b). Cocoa has a potent antioxidant capacity compared to products
traditionally considered high in antioxidants (Lee et al., 2003; Vinson et al., 2006).
Flavonoids act as antioxidants by directly neutralizing free radicals, chelating Fe2+ and Cu+
which enhance highly aggressive ROS, inhibiting xanthine oxidase that is responsible for
ROS production, and up-regulating or protecting antioxidant defense (Cotelle, 2001).
Epicatechin and catechin are very effective in neutralizing several types of free radicals
(Hatano et al., 2002; Yilmaz et al., 2004). Procyanidins account for the highest percentage of
antioxidants in cocoa products (Gu et al., 2006) and they also scavenge radicals with an
activity that is proportional to the number of monomeric units they contain (Counet et al.,
Effect of a Diet Rich in Cocoa Flavonoids… 215
Eight-week-old female Wistar rats were obtained from Harlan (Barcelona, Spain). Rats
were housed 3 per cage in controlled conditions of temperature and humidity in a 12:12
light:dark cycle. Rats had free access to food (chow ref. 2014, Harlan Teklad, Madison, WI,
USA) and water. Animals were randomly distributed in 3-4 experimental groups in each
experimental design (n = 8-10/group). Two of them were daily administered, by oral gavage,
with cocoa in mineral water at doses of 2.4 g/kg/day and 4.8 g/kg/day for 7 days. We used
Natural Forastero cocoa (Nutrexpa, Barcelona, Spain) containing 21.2 mg of total phenols/g
according to the Folin-Ciocalteu method (Singleton et al., 1999). The remaining animals
received the same volume of vehicle (mineral water). Handling was done in the same time
range to avoid the influence of biological rhythms. At the end of the study rats were sacrificed
by CO2 inhalation. Studies were performed in accordance with the institutional guidelines for
216 M. Castell, A. Franch, S. Ramos-Romero et al.
the care and use of laboratory animals established by the Ethical Committee for Animal
Experimentation at the University of Barcelona and approved by the Catalonian Government.
After 7 days of cocoa or vehicle administration p.o., rats were anaesthetized with
ketamine/xylacine (i.m., 90 mg/kg and 10 mg/kg, respectively) to obtain peritoneal
macrophages. 40 mL ice-cold sterile phosphate buffer solution (PBS) pH 7.2 was injected to
peritoneal cavity. Abdominal massages were immediately performed to induce cell migration.
Cell suspension was aspirated, centrifuged (170 g, 5 min, 4 ºC) and resuspended in cold
DMEM+GlutaMAX media (Invitrogen, Paisley, UK) containing 10% fetal bovine serum
(PAA, Pashing, Austria), 100 IU/mL streptomycin-penicillin (Sigma-Aldrich, St Louis, MO,
USA) (DMEM-FBS). Cell count and viability was determined by double staining with
acridine orange and ethidium bromide (Sigma) followed by fluorescence light microscopical
analysis. Cells were plated and cultured in different conditions according to the assay.
To determine the effects of cocoa on ROS production, peritoneal macrophages (25 x 103
cells/100 µL in DMEM-FBS) were plated in 96 well black plates (Corning Inc, NY, USA)
and allowed to attach overnight (37 ºC, 5% CO2). Macrophages were washed once with warm
RPMI-1640 medium without phenol red (Sigma) containing 100 IU/mL streptomycin-
penicillin and incubated with 20 µmol/L of reduced 2’,7’-dichlorofluorescein diacetate
(H2DCF-DA, Invitrogen) probe for 30 min at 37 ºC. H2DCF-DA diffuses through the cell
membrane and is enzymatically hydrolyzed by intracellular esterases to form non-fluorescent
2’,7’-dichlorofluorescein (H2DCF) which is oxidized by ROS to a fluorescent compound
(DCF). Thus, DCF fluorescence intensity is proportional to intracellular ROS production.
Fluorescence was measured every 30 min by fluorometry (excitation 538 nm, emission 485
nm) up to 3.5 h. For each animal, background from corresponding wells without fluorescent
probe was subtracted.
Immediately after isolation, macrophages were plated in 12-well flat-bottom plate (TPP,
Trasadingen, Switzerland) at 1.2 x 106/mL in DMEM-FBS (37 ºC, 5% CO2) overnight to
allow macrophage adhesion. Non-adherent cells were removed by washing three times with
warm sterile PBS. The attached macrophages were stimulated by addition of 1 µg/mL
lypopolysaccharide (LPS) from E.coli O55:B5 (Sigma). Supernatants were collected for
quantification of TNFα after 6 h and IL-6 and NO after 24 h. Supernatants were stored at
-80 ºC until evaluation. Cells were harvested to determine cell viability. The concentration of
TNFα and IL-6 in supernatants was quantified using rat ELISA sets from BD Pharmingen
(Erembodegen, Belgium), following the manufacturer’s instructions.
Effect of a Diet Rich in Cocoa Flavonoids… 217
Stable end product of NO, NO2-, was quantified by a modification of Griess reaction.
Briefly, macrophage supernatants (100 µL) were mixed with 60 µL sulphanilamide 1% (in
1.2 N HCl) and 60 µL N-(1-naphthyl)ethylene-diamine dihydrochloride 0.3% (in distilled
water) for 10 min at room temperature. Absorbance was read spectrophotometrically at
540 nm. The concentration of NO2- was calculated using known concentration of NaNO2.
Paw volume was measured by using a water plethysmometer (UGO Basile, Comerlo,
VA, Italy). Left and right hind paws were measured just before the induction (time 0). After
carrageenin injection, paw volumes were quantified at 30 min and every hour until 6 h. In the
other 4 experimental models, the measurements were performed each 15 min during the first
hour, and each 30 min up to 2 h. All determinations were performed in a blinded manner. Paw
volumes were expressed as percentage of increase with respect to time 0. Area under curve
(AUC) was calculated between time 0 and the end of the inflammatory period evaluation.
2.7. Statistics
The software package SPSS 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical
analysis. Conventional one-way ANOVA was performed, considering the experimental group
as independent variable. When treatment had a significant effect on dependent variable,
Scheffe’s test was applied. Significant differences were accepted when P<0.05.
3. RESULTS
3.1. Peritoneal Macrophages Viability
Peritoneal macrophages were obtained from rats after cocoa intake (2.4 or 4.8 g/kg/day)
or vehicle for 7 days. Cells were 98% viable when isolated. After overnight culture and
washing, some cells were LPS-stimulated and 6 h later, they showed a viability of about 40%
218 M. Castell, A. Franch, S. Ramos-Romero et al.
whereas non-stimulated cultures were ~50% viable. However, 24 h after LPS stimulation,
macrophage viability reached ~70% and 75% in LPS-stimulated and non-stimulated
macrophages, respectively. There were no differences among cells obtained from both cocoa-
administered rats and those from reference animals.
ROS production from peritoneal macrophages increased progressively along the 3.5 h
assay (Figure 1A). Cells obtained from 2.4 g/kg cocoa animals produced the same ROS levels
as reference macrophages. Macrophages isolated from animals that received 4.8 g/kg/day of
cocoa synthesized lower ROS than reference cells already at 0.5 h and all along the studied
period. Differences between both groups were higher as later measurements were made but
they did not reach statistically significant results because of the high variability.
NO production was detected in macrophage culture medium after 24 h of LPS-
stimulation or in resting conditions (Figure 1B).
Macrophages obtained from rats that received 2.4 g/kg/day of cocoa showed NO levels
similar to those from reference group, both in LPS-stimulation and in resting conditions.
Nevertheless, NO secretion by macrophages isolated from animals with an intake of
4.8 g/kg/day of cocoa was lower than that quantified in the reference group in any culture
condition (P<0.05, Figure 1B).
Figure 1. ROS and NO production by peritoneal macrophages. Time-course of ROS production from
peritoneal macrophages (A) was determined by means of DCF assay. NO cell production, quantified as
NO2- concentration (B), was measured by modified Griess assay in LPS-stimulated and resting
macrophages. Values are summarized as mean ± SEM (n = 8-9). * P<0.05 compared with reference
group.
Effect of a Diet Rich in Cocoa Flavonoids… 219
Figure 2. TNFα and IL-6 secretion from peritoneal macrophages. TNFα (A) and IL-6 (B) concentration
(ng/mL) in cell culture supernatants was determined by means of ELISA assay. Values are summarized
as mean ± SEM (n = 8-9). * P<0.05 compared with reference group. φ P<0.05 compared with 2.4 g/kg
cocoa group.
220 M. Castell, A. Franch, S. Ramos-Romero et al.
Figure 4. Time-course of paw edema in acute inflammatory models. Time-course of histamine- (A),
serotonin- (B), bradykinin- (C), and PGE2-induced acute inflammation (D) in the studied groups.
Values are summarized as mean ± SEM (n = 8-10). * P<0.05 compared with reference group.
Cocoa-enriched diet did not protect from the development of histamine-, serotonin- and
PGE2-induced paw edema. Nevertheless, a significant reduction of paw volume increase was
detected in 2.4 g/kg cocoa group in the bradykinin model. These animals showed lower
inflammation at 15 min post-injection (P<0.05) and during all studied period (P<0.01). Daily
intake of 2.4 g/kg cocoa reduced up to 28% the AUC of hind paw volume evolution (P<0.05,
data not shown). Indomethacin did not show anti-inflammatory effects in these models of
acute inflammation (data not shown).
DISCUSSION
This study shows the in vivo anti-inflammatory power of a high cocoa intake for a week.
Although several studies demonstrate the regulatory effect of cocoa flavonoids in vitro on
222 M. Castell, A. Franch, S. Ramos-Romero et al.
cells under inflammatory stimulus (Mao et al., 2000; Ono et al., 2003; Ramiro et al., 2005),
there are few evidences of the effect of cocoa on inflammatory response in physiological
conditions. Here we show two in vivo evidences that allow suggesting the inflammation
inhibition by a cocoa diet.
The first part of this study shows that a high cocoa intake for a week reduces the
inflammatory potential of macrophages. During an inflammatory response, macrophages are
crucial cells participating in the secretion of mediators that eventually induce vasodilatation,
vascular permeability increase and leukocyte migration (Medzhitov, 2008). Moreover,
macrophages produce a battery of reactive oxygen, nitrogen and halogen species which are
proposed to cause damage to surrounding tissues (Son et al., 2008). In the present study,
macrophages from animals that had taken a high dose of cocoa produced lower levels of
reactive nitrogen species and also had a tendency to synthesize lower reactive oxygen species.
These results agree with in vitro studies showing that cells from different origins treated with
cocoa fractions or flavonoids alone decrease the production of ROS in a dose-dependent
manner (Sanbongi et al., 1997, Erlejman et al., 2006; Granado-Serrano et al., 2007; Ramiro-
Puig et al., 2009a). Moreover, cocoa fractions are reported to reduce the levels of NO when
produced in inflammatory conditions (Ono et al., 2003; Lyu et al., 2005; Ramiro et al., 2005).
These results seem to be contradictory with the effects of cocoa flavonoids in vascular tissue
where they are reported to promote NO bioactivity and then to cause vasodilatation and
decrease blood pressure (Sies et al., 2005; Taubert et al., 2007). These opposite effects seem
to be related to the enzyme isoform involved in NO synthesis: eNOS in the endothelial
region, or iNOS after an aggressive stimulus (Karim et al., 2000; Ono et al., 2003). Therefore,
cocoa compounds would have contrary effects depending on these enzyme isoforms.
In a similar way, macrophages isolated from animals that had taken a high dose of cocoa
for a week showed a lower ability to secrete two essential cytokines in the inflammatory
process, TNFα and IL-6. These results agree with those previously obtained after adding a
cocoa extract on a macrophage cell line (Ramiro et al., 2005), although there are controversial
results when cocoa flavonoid fractions were used on blood mononuclear cells (Mao et al.,
2000; Kenny et al., 2007). In any case, an important difference between these in vitro studies
and those showed here consists in the compounds that achieve cells. In a more physiological
approach, the present study suggests that metabolites derived from cocoa absorbed fractions
have anti-inflammatory properties. How macrophages down-regulate their inflammatory
response in the presence of cocoa metabolites remains to be established. However, some in
vitro studies show that flavonoids such as epicatechin, catechin, dimeric procyanidins and
quercetin can modify the NF-κB pathway (Mackenzie et al., 2004; Comalada et al., 2005)
involved in the production of inflammatory products. Therefore, it can be suggested that the
cocoa absorbed fraction, flavonoids or even other compounds, can also interact with this
transduction pathway.
After demonstrating the effect of cocoa in reducing some inflammatory mediators ex
vivo, the second part of this study was focused on ascertaining whether a cocoa enriched diet
was able to directly modulate a local inflammation in vivo. Specifically, we examined the
effect of cocoa on the local inflammatory response induced by carrageenin in the rat hind paw
(Winter et al., 1962). This model is widely applied for the screening of anti-inflammatory
drugs which provokes a progressive local edema during 4–6 h, that remains even up to 24 h.
Carrageenin-induced edema is accompanied with prostanoids and pro-inflammatory cytokines
Effect of a Diet Rich in Cocoa Flavonoids… 223
pain (Marceau et al., 2004). These actions would not be affected by a cocoa diet and then
would explain the mild effect of cocoa on the bradykinin model. In addition, the beneficial
properties of cocoa on this model were not observed at the highest dose. This lack of effect
could be explained by the vasodilator consequences of cocoa intake, which at high
concentration would predominate over the antagonistic action of flavonoids on bradykinin.
Therefore, from the experimental models induced by single inflammatory mediators, it
could be suggested that the anti-inflammatory effect of 2.4 g/kg/day cocoa was due at least
partially by regulating bradykinin actions. This effect could explain the inhibition observed
with this dose during the first hour in the carrageenin model. Later, the same dose would not
be able to counteract the macrophage activation phase, which agrees with results from
peritoneal macrophages. On the contrary, higher doses of cocoa inhibit carrageenin-induced
edema longer, which could be the result of the down-regulation of mediators produced by
macrophages as reactive oxygen and nitrogen species and cytokines.
In summary, a high intake of cocoa could produce anti-inflammatory effects in vivo, as
shown in vitro. Although it remains to be ascertained the precise mechanism of action of
cocoa and its effectiveness in other inflammatory processes, cocoa seems a good candidate to
be considered as a functional food.
ACKNOWLEDGMENTS
S.R. is the recipient of fellowships from the Ministerio de Educación y Ciencia (BES-
2006-13640). The present study was supported by the Ministerio de Educación y Ciencia,
Spain (AGL2005-002823) and from SGR 2005-0083 of the Generalitat de Catalunya.
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In: Flavonoids: Biosynthesis, Biological Effects… ISBN: 978-1-60741-622-7
Editor: Raymond B. Keller © 2009 Nova Science Publishers, Inc.
Chapter 7
ABSTRACT
The biological activity of flavonoids was first recognized when the antiestrogenic
principle present in red clover that caused infertility in sheep in Western Australia was
discovered. These adverse effects of flavonoids placed these substances in the class of
endocrine-disrupting chemicals. On the other hand, flavonoids are recently claimed to
prevent several cancer types and to reduce incidence of cardiovascular diseases,
osteoporosis, neurodegenerative diseases, as well as chronic and acute inflammation.
Despite these controversial effects, a huge number of plant extracts or mixtures,
containing varying amounts of isolated flavonoids, are commercially available on the
market as dietary supplements and healthy products. The commercial success of these
supplements is evident, even though the activity and mechanisms of flavonoid action are
still unclear.
Owing to their chemical structure, the most obvious feature of flavonoids is their
ability to quench free radicals. However, in the last few years many exciting new
indication in elucidating the mechanisms of flavonoid actions have been published.
Flavonoids inhibit several signal transduction-involved kinases and affect protein
functions via competitive or allosteric interactions. Among others, flavonoids interact
with and affect the cellular responses mediated by estrogen receptors (ERα and ERβ). In
particular, our recent data indicate that some flavonoids (i.e., naringenin and quercetin)
decouple specific ERα action mechanisms, important for cell proliferation, driving cells
to the apoptosis. Therefore, distinct complex mechanisms of actions, possibly interacting
one another, for nutritional molecules on cell signalling and response can be
hypothesized.
∗
Tel. 0039-06-57336345; fax 0039-06-57336321. e-mail address: m.marino@uniroma3.it (M. Marino)
232 Maria Marino and Pamela Bulzomi
1. INTRODUCTION
Flavonoids, phenylbenzo-pyrones (phenylchromones), are a large group of non nutrient
compounds naturally produced from plants as part of their defence mechanisms against
stresses of different origins. They are present in all terrestrial vascular plants; whereas, in
mammals, flavonoids occur only through dietary intake (Birt et al., 2001). Flavonoids, have
an assortment of structures based on a common three-ring nucleus (Middleton et al., 2000) in
which primary substituents (eg, hydroxyl, methoxyl, or glycosyl groups) can be further
substituted (e.g., additionally glycosylated or acylated) sometimes yielding highly complex
structures (Cheynier, 2005) (Figure 1). More than 4,000 different flavonoids, have been
described and categorized into 6 subclasses as a function of the type of heterocycle involved:
flavonols, flavones, flavanols, flavanonols, flavanones, and isoflavones (Figure 1) (Birt et al.,
2001; Manach et al., 2004).
First recognized in ’40 as the antiestrogenic principle present in red clover that caused
infertility in sheep in Western Australia (Bennetts et al., 1946; Galluzzo and Marino, 2006),
more recently, the use of flavonoids to curb menopausal symptoms and provide a “natural”
and presumably cancer risk-free estrogenic replacement (Ling et al., 2004) has become
popular in Western countries.
Thus, a huge number of preparations are now commercially available on the market as
health food products. As dietary supplements they are obtainable as plant extracts or
mixtures, containing varying amounts of isolated or concentrate flavonoids in bakery, dairy,
infant formulas (Tomar and Shiao, 2008). The commercial success of these supplements is
evident and the consumption of these compounds in Western countries is increasing even if
different and opposite flavonoid effects have been reported.
In particular, epidemiological data show that in pre-menopausal women, assuming daily
soy, follicle stimulating and luteinizing hormone levels significantly decreased, increasing
menstrual cycle length (Jacobs and Lewis, 2002). Hyperplasia of mammary glands in both
sexes, aberrant or delayed spermatogenesis, histological changes in the vagina and ovary,
mineralization of renal tubules in males, modulation of natural killer cell activity, and
myelotoxicity had been observed in rats exposed to isoflavone genistein through placental
transfer or lactational exposure, or ingestion (Flynn et al., 2000; Delclos et al., 2001; Guo et
al., 2005; Doerge et al., 2006). On the other hand, a positive association between the
increased intake of phytoestrogen and reduced amount of neurodegenerative diseases,
improvement of cognition and learning (Kirk et al., 1998), and fewer tendency to osteoporosis
(Zhang et al., 2003; Adlercreutz et al., 2004) have also been reported. In addition, diets rich in
flavonoids also lead to lower serum cholesterol levels, low-density lipoproteins, and
triglycerides (Delclos et al., 2001), thus reducing the incidence of cardiovascular diseases
(Doerge et al., 2006). Finally, the close relationship between flavonoids and cancer is
suggested by the large variation in rates of specific cancers in different countries and by the
spectacular changes observed in the incidence of cancer in migrating populations (Guo et al.,
2005; Béliveau and Gingras, 2007; Benavente-García and Castillo, 2008). These observations
are strengthened by many experimental data obtained from studies using cellular and animal
models (Caltagirone et al., 2000; Fenton and Hord, 2004; Albini et al., 2005; Béliveau and
Gingras, 2007; Espìn et al., 2007; Benavente-García and Castillo, 2008).
Flavonoids have been considered able to modulate this wide spectrum of responses due to
their chemical structure compatible with putative antioxidant properties which can interact
with reactive oxygen-nitrogen species (RONS)-mediated intracellular signaling (Virgili and
Marino, 2008). However, different cellular effects not directly related to flavonoid antioxidant
capacity have been recently reported. Flavonoids could modulate the activity of several
kinases and could affect protein functions (e.g. receptors) via competitive or allosteric
interactions. Thus, flavonoids should be considered pleiotropic substances which possess
distinct mechanisms of action possibly interacting each other.
Aim of this review is to provide an update about mechanisms by which flavonoids play a
role in cellular response and in preventing cancer.
consequence of RONS action is the damage of membrane lipids, proteins, and DNA with the
subsequent onset of various diseases (Moskaug et al., 2005).
RONS, either directly or indirectly, regulates the activity of some of the most well-known
signaling enzymes including guanylyl cyclase, phospholipase C, phospholipase A2,
phospholipase D, activating protein-1 (AP-1), nuclear factor κB (NF-κB), insulin receptor, c-
Src, Jun N-terminal kinase, and p38 kinase (MAPK) (Hehner et al., 2000). Moreover, a
significant inhibition of phosphatase activity, paralleled by the net increase of
phosphorylation level, has been reported (O'Loghlen et al., 2003, Hao et al., 2006).
Due to their chemical structure, flavonoids are able to quench free radicals by forming
resonance-stabilized phenoxyl radicals in vitro, in cell cultures, and in cell free systems
(Hanasaki et al., 1994, Ursini et al., 1994, Birt et al., 2001). In addition, these compounds
have been considered able to impair the RONS-mediated intracellular signaling by directly
inhibiting the involved enzymes or by chelating trace elements involved in free radical
production or up-regulating the antioxidant cellular response (Surh, 2003, Lee et al., 2005,
Chiang et al., 2006, Kweon et al., 2006). Thus, an high intake of flavonoids should be
associated with a reduced risk of degenerative diseases such as cardiovascular disease and
cancer. Experimental data have shown that flavonoids block the progression of latent
microtumors (Béliveau and Gingras, 2007) and this effect could be elicited via the modulation
of the enzymatic systems responsible for neutralizing free radicals (Conney, 2003; Ioannides
and Lewis 2004) and/or by directly inducing cancer cell death by apoptosis. For example,
phenethyl isothiocyanate from cruciferous vegetables, curcumin from turmeric, resveratrol
from grapes, and naringenin from oranges have all been shown to possess strong pro-
apoptotic activity against cells isolated from a variety of tumors (Totta et al., 2004;
Karunagaran et al., 2005; Totta et al., 2005). Thus, the antioxidant activity of flavonoids
could represent the close relationship between flavonoids and cancer chemoprevention.
However, flavonoids also possess pro-oxidant properties which could contribute to their
anti-cancer activity (Galati et al., 2000). Flavonoid pro-oxidant activity seems to be involved
in the inhibition of mitochondrial respiration and is related to their in vitro ability to undergo
to auto-oxidation to produce superoxide anions. On the other hand, transition metals, which
catalyze auto-oxidation, result linked to proteins in vivo and it is unlikely they could
significantly participate in the auto-oxidation of polyphenols. As alternative mechanism
flavonoids pro-oxidant activity could be dependent from the activity of peroxidases (O’Brien,
2000). As an example, the apigenin in the presence of myeloperoxidase forms a peroxyl
radical in vitro (Galati et al., 2001) which leads to the intracellular production of ROS and
contributes to the apoptotic and necrotic cell death (Wang et al., 1999; Morrissey et al., 2005;
Vargo et al., 2006; Miyoshi et al., 2007). Baicalin-apoptosis induction is also accompanied
with the generation of intracellular ROS and the increase of the cytochrome c release (Ueda et
al., 2002). Cytochrome c release and mitochondrial transmembrane potential disruption
precedes the apoptosoma activation in MCF-7 cell treated with 200μM of the stilbene trans-
resveratrol (Filomeni et al., 2007). Contrasting results have been obtained treating cells with
quercetin. This flavonoid possess an high antioxidant activity preventing the H2O2-induced
ROS production in a dose-dependent manner not related with its pro-apoptotic effect. In
addition, the cell treatment with 50 μM quercetin induces an increase of ROS production in a
cell context dependent way (Galluzzo et al., 2008 and literature cited therein). Thus, other
action mechanism(s) independent from antioxidant/pro-oxidant effects of flavonoids should
Mechanisms at the Root of Flavonoid Action in Cancer 235
Figure 2. NRs share an evolutionarily conserved structure consisting of the high variable N-amino-
terminal region involved in transactivation (A and B domains), the conserved DNA binding region
(DBD, C domain), the hinge region, the ligand binding domain (LBD; E domain), and the C-terminal
region (F domain). For details, see text.
Mechanisms at the Root of Flavonoid Action in Cancer 237
This data is consistent with the hypothesis that soy isoflavones improve lipid metabolism
and have an antidiabetic effect by activating PPAR receptors and is in agreement with
observations in humans treated with antidiabetic PPARα agonists (e.g. GW501516) used to
treat hyperlipidemia and type 2 diabetes (Elisaf et al., 2002).
Mixtures of isoflavones and isolated isoflavones have been reported to induce PXR
transcriptional activity (Ricketts et al., 2005) which has been shown to be activated by a
chemically and structurally diverse set of xenobiotic and endogenous compounds and to
regulate gene expression pathways involved in metabolism and transport of these same
classes of compounds (Moore and Kliewer, 2000; Wang et al., 2007). Notably, PXR has been
shown to directly regulate the cytochrome P450 3A gene, a phase I drug metabolism gene,
whose product is responsible for the metabolism of drugs (Moore and Kliewer, 2000; Wang
et al., 2007). Other authors reported that genistein, formononetin, kaempferol, and apigenin
did not exhibit PXR ligand activity (Mnif et al., 2007). Thus, at present the contribution of
PXR signaling to the flavonoid effects on human health remains elusive. Contrasting data
have also been reported about flavonoids effects on AhR. Quercetin, galangin, diosmin, and
diosmetin increase the expression of phase I enzymes, important for the prevention of cancer,
by binding to AhR receptor (Kang et al., 1999).
Computer docking analyses and mammalian two-hybrid experiments indicate that
isoflavones (i.e., genistein, daidzein, and biochanin A) and one flavone (trihydroxyflavone)
are relatively poor ligands of estrogen related receptor γ (ERRγ, NR3) but they can act as
agonists of ERRα and ERRβ activity (Suetsugi et al., 2003).
membrane, association to caveolin-1, and which accounts for the ability of E2 to activate
different signaling pathways (Acconcia et al., 2005a). The Cys399 residue present in the LBD
of ERβ is also subjected to S-palmitoylation (Galluzzo et al., 2007) indicating that a similar
mechanism also works for ERβ localization to the plasma membrane and association to
caveolin-1. Thus, ERα and ERβ have to be considered as a population of protein(s) which
localization in the cell can dynamically change, shuttling from membrane to cytosol and to
the nucleus on the dependence of E2 binding (Acconcia et al., 2005a; Levin, 2005). As a
consequence, rapid and more prolonged E2 actions could be more finely coordinated. The
physiological significance of ERs-dependent rapid pathways is quite clarified, at least for
some E2 target tissues. The mechanism by which E2 exerts proliferative properties has been
assumed to be exclusively mediated by ERα-induced rapid membrane-starting actions
(Marino et al., 2005; Ascenzi et al., 2006), whereas E2 induces cell death through ERβ non-
genomic signaling (Acconcia et al., 2005b). In the nervous system, E2 influences neural
functions (e.g., cognition, behavior, stress responses, and reproduction) in part inducing such
rapid responses (Farach-Carson and Davis, 2003). In the liver, rapid E2-induced signals are
deeply linked to the expression of LDL-receptor and to the decreased cholesterol-LDL levels
in the plasma (Distefano et al., 2002). An important mode of E2-mediated atheroprotection is
linked to E2 capability to rapidly activate endothelial NOS and NO production (Chambliss et
al., 2002).
These rapid effects (E2 extranuclear signals) include the activation of mitogen-activated
protein kinase (MAPK) (i.e., p38, extranuclear regulated kinases [ERK]), PI3K, signal
transducer and activator of transcription, epidermal growth factor receptor, Src kinase, Shc
kinase, protein kinase C, adenylate cyclase, GTP-binding proteins, and NOS (Dang and
Lowik, 2005). Microarray analysis of gene expression in vascular endothelial cells treated
with E2 for 40 min showed that 250 genes were up-regulated; this could be prevented by
Ly294002, a PI3K inhibitor. Interestingly, the transcriptional activity of the E2-ERα complex
could be inhibited by pre-treating cells with PD98059 and U0126, two ERK inhibitors
(Ascenzi et al., 2006). These findings support the idea that E2-induced rapid signals synergize
with genomic events to maintain the pleiotropic hormone effects in the body (Galluzzo and
Marino, 2006).
A plethora of papers indicates the ability of flavonoids to bind both ER isoforms
maintaining the ERs gene transcriptional ability, nevertheless several epidemiological and
experimental data show that flavonoid effects can be both estrogen mimetic and
antiestrogenic. Several groups have demonstrated that flavonoid affinity to ERs is lower than
E2 (Kuiper et al., 1997). Competition binding studies confirm that nutritional molecules (e.g.,
genistein, coumestrol, daidzein, and equol) show a distinct preference for ERβ (Kuiper et al.,
1997; Mueller et al., 2004; Escande et al., 2006), although the prenylated chalcone occurring
in hops, 8-prenylnaringenin, has been found to be a potent ERα agonist, but a weak agonist of
ERβ in E2 competition assays (Stevens and Page, 2004). Phytochemicals as the isoflavonoids
daidzein and genistein, the flavanone naringenin, and the flavonol quercetin increase the
activity of ERE-luciferase reporter gene construct in cells expressing ERα or ERβ (Mueller,
2002; Totta et al., 2004; Virgili et al., 2004; Totta et al., 2005), but impair ERα interaction
with Sp-1 and AP-1 (Peach et al., 1997; Liu et al., 2002; Virgili et al., 2004). Cluster analysis
of DNA microarray in MCF-7 cells show a very similar profiles between estrogen responding
genes and 10 μM genistein (Terasaka et al., 2004) while the expression of only five genes is
affected by daidzein with respect to E2 in TM4 Sertoli cells. These five genes were related to
Mechanisms at the Root of Flavonoid Action in Cancer 239
cell signaling, cell proliferation, and apoptosis, suggesting a possible correlation with the
inhibition of cell viability reported after treatment with daidzein (Adachi et al., 2005).
The capability of flavonoids to influence E2 rapid actions in both reproductive and non-
reproductive E2-target tissues and how such effects may impact the normal development and
physiological properties of cells largely have not been tackled until very recently (Somjen,
2005; Watson, 2005). In fact, scarce information is available on the non genomic signal
transduction pathways activated after the formation of flavonoids:ERα and flavonoids:ERβ
complexes. Recent evidence favors the idea that besides coactivator association, the ER-LBD
is essential and sufficient also for activation of rapid E2-induced signals (Marino et al., 2005).
Thus, it is possible that flavonoids could induce different conformational changes of ER, also
precluding the activation of rapid signaling cascades (Galluzzo et al., 2008). As support of
this hypothesis our group have recently demonstrated that quercetin and naringenin hamper
ERα-mediated rapid activation of signaling kinases (i.e., ERK/ MAPK and PI3K/AKT) and
cyclin D1 transcription only when HeLa cells, devoid of any ER isoforms, were transiently
transfected with a human ERα expression vector (Virgili et al., 2004). In particular,
naringenin, inducing conformational changes in ER, provokes ERα depalmitoylation faster
than E2, which results in receptor rapid dissociation from caveolin-1, impairing ERα binding
to molecular adaptor and signaling proteins (e.g., modulator of non genomic actions of the
ER, c-Src) involved in the activation of the mitogenic signaling cascades (i.e., ERK/MAPK
and PI3K/AKT) (Galluzzo et al., 2008). Moreover, naringenin induces the ERα-dependent,
but palmitoylation-independent, activation of p38/MAPK, which in turn is responsible for
naringenin-mediated antiproliferative effects in cancer cells. Naringenin, decoupling ERα
action mechanisms, prevents the activation ERK/MAPK and PI3K/AKT signal transduction
pathways thus, drives cells to apoptosis (Galluzzo et al., 2008). On the other hand, naringenin
does not impair the ERα-mediated transcriptional activity of an ERE-containing promoter
(Totta et al., 2004; Virgili et al., 2004). As a whole, this flavanone modulates specific ERα
mechanisms and can be considered as ‘mechanism-specific ligands of ER’ (Totta et al.,
2004).
In the same cell system, we recently demonstrated that quercetin activates the rapid ERα-
dependent phosphorylation of p38/MAPK and, in turn, the induction of a proapoptotic
cascade (i.e., caspase-3 activation and PARP cleavage) (Galluzzo et al., 2008a). This result
proves that quercetin- and naringenin-induced apoptosis in cancer cells depends on the
flavonoid interference with ERα-mediated rapid actions suggesting a role of endocrine
disruptor for these flavonoids (Galluzzo et al 2008a).
As previously described, E2 mediates a wide variety of complex biological processes
including skeletal muscle differentiation via ERα-dependent signals (Marino, personal
communication). Our recent data show that Nar stimulation of rat skeletal muscle cells (L6),
decoupling ERα mechanism of action, impedes the E2-dependent differentiation further
sustaining the antiestrogenic role played by flavonoids (Marino, personal communication).
Thus, flavonoids have a very complex spectrum of activities: they can function as
mechanism-specific ligands of ERα (Totta et al., 2004) due to their ability to decouple ERα
activities, eliciting estrogenic or antiestrogenic effects downstream of these pathways
(Galluzzo et al., 2008).
240 Maria Marino and Pamela Bulzomi
CONCLUSIONS
Interest on dietary compounds has evolved since their therapeutic properties has been
discovered. Flavonoids are documented to play a major role amongst the hormonally agents
in food, nonetheless the importance of their role in human health has not yet unequivocally
established.
Flavonoids have a chemical structure compatible with a strong putative antioxidant.
Neverthless, flavonoids antioxidant/pro-oxidant properties could be considered a simplified
approach to the function of molecules of nutritional interest due to the fact that their
antioxidant and/or pro-oxidant capacities are chemical properties which are not necessarily
associated to an equivalent biological function (Virgili and Marino, 2008). Remarkably,
flavonoids have also a strong affinity for protein so they can modulate cellular function
inhibiting or modulating protein functions (Figure 3).
Normally, human flavonoid plasma concentrations are in the low nanomolar range, but
upon flavonoid supplementation they may increase to the high nanomolar or low micromolar
range (Boots et al., 2008).
Thus, flavonoids do not appear to be present in the circulation at high enough
concentrations to contribute significantly to total antioxidant capacity (concentration of
circulating endogenous antioxidant, ascorbate or urate, has been estimated to be in the range
of 159-380 μmol/l for a normal individual) (Stevenson and Hurst, 2007) or inhibition kinases
activity.
Figure 3. Schematic model illustrating the multiple effects of flavonoids on cell functions.
Mechanisms at the Root of Flavonoid Action in Cancer 241
ACKNOWLEDGMENTS
The Authors wish to thank past and present members of their laboratories who
contributed to the ideas presented here through data and discussions.
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In: Flavonoids: Biosynthesis, Biological Effects… ISBN: 978-1-60741-622-7
Editor: Raymond B. Keller © 2009 Nova Science Publishers, Inc.
Chapter 8
ANTIOPHIDIAN MECHANISMS
OF MEDICINAL PLANTS
ABSTRACT
Vegetal extracts usually have a large diversity of bioactive compounds showing
several pharmacological activities, including antiophidian properties. In this study, both
coumarin and tannic acid (100 µg/mL) showed no changes in the basal response of
twitches in mouse nerve phrenic diaphragm preparations. In opposite, Crotalus durissus
terrificus (Cdt 15 µg/mL) or Bothrops jararacussu (Bjssu 40 µg/mL) venoms caused
irreversible neuromuscular blockade. Tannic acid (preincubated with the venoms), but
not coumarin, was able to significantly inhibit (p<0.05) the impairment of the muscle
strength induced by Cdt (88 ± 8%) and Bjssu (79 ± 7.5%), respectively. A remarkable
precipitation was observed when the venoms were preincubated with tannic acid, but not
with coumarin. Plathymenia reticulata is a good source of tannins and flavonoids
whereas Mikania laevigata contain high amounts of coumarin. P. reticulata (PrHE, 0.06
mg/mL) and M. laevigata (MlHE, 1 mg/mL) hydroalcoholic extracts were assayed with
or without Bjssu or Cdt venoms. Both PrHE and MlHE showed protection against Bjssu
(79.3 ± 9.5% and 65 ± 8%, respectively) and Cdt (73.2 ± 6.7% and 95 ± 7%,
respectively) neuromuscular blockade. In order to observe if the protective mechanism
could be induced by protein precipitation, tannins were eliminated from both extracts and
∗
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250 Rafael da Silva Melo, Nicole Moreira Farrapo et al.
the assay was repeated. MlHE protected against the blockade induced by Bjssu (57.2 ±
6.7%), but not against Cdt. We concluded that plants containing tannins could induce the
precipitation of venoms’ proteins and plants containing coumarin showed activity against
Bothrops venoms, but not against Crotalus venoms. We also concluded that the use of
isolated bioactive compounds could not represent the better strategy against ophidian
venoms, since the purification may exclude some bioactive components resulting in a
loss of antivenom activity. In addition, M. laevigata showed better antiophidian activity
than P. reticulata.
INTRODUCTION
The use of medicinal plants has been practiced for many generations. In addition, plants
had contributed for the development of many valuable substances such as morphine, the
principal alkaloid in opium and the prototype opiate analgesic and narcotic; vincristine, the
antitumor alkaloid isolated from Vinca Rosea; rutin, a flavonol glycoside found in many
plants, used therapeutically to decrease capillary fragility [4].
The animal kingdom constitutes another interesting source of investigation, mainly
venomous animals, first due to its pathological effects caused by envenomation and second by
the therapeutical possibilities of their constituents. For example, bradykinin, a nonapeptide
messenger enzymatically produced from kallidin in the blood, is derived from Bothrops
jararaca venom [21].
Many people have been seeking complementary and alternative medicines as an adjunct
to conventional therapies. There is a renewed interest in the therapeutic potential of venoms
from bees, snakes and scorpions or sea-anemones toxins [23]. Salmosin, a desintegrin derived
from snake venom that contains the Arg-Gly-Asp (RGD) sequence, was reported to be both
antiangiogenic and anti-tumorigenic [18].
The natural resources are the largest reservoir of drugs [29], and the investigation of
substances with therapeutic effects has been performed by isolation, extraction and/or
purification of new compounds of vegetable origin [1]. Nowadays, there are many
sophisticated - but yet very expensive, laborious and long-time demanding – methods to
obtain these new compounds from plant or animal origin.
One of the most used experimental models to test pharmacological and antivenom
properties of new compounds is the nerve phrenic diaphragm preparations isolated from rats
[3], which was also modified for mice. Anatomically and physiologically this preparation
represents the nerve-muscle synapse and the muscular contraction, respectively.
Pharmacological effects can be showed by blockade, facilitation or contracture, among other
possibilities. B. jararacussu venom, for example, causes both neuromuscular blockade and
severe local myonecrosis.
Mikania laevigata, popularly known as “guaco”, is related to Mikania glomerata
Sprengel, being both used to treat respiratory diseases. Both have pharmacological activities
attributed to coumarin [5, 12, 20, 24]. The main difference between the two species is the
flowering period: M. laevigata flourishes in September and M. glomerata in January [24].
Antiophidian Mechanisms of Medicinal Plants 251
Some authors have also attributed an antiophidian property against brazilian snake venoms to
M. glomerata [20, 28], but this property was not studied in M. laevigata.
Plathymenia reticulata Benth, popularly known as “vinhático”, is a plant from Brazilian
“cerrado” that has anti-inflammatory properties, and a previous phytochemical study
identified tannins and flavonoids as its principal constituents [11].
The aim of the present study was to investigate the neutralizing ability of two commercial
phytochemicals (tannic acid and coumarin) against the neuromuscular blockade induced by
two crude snake venoms - Bothrops jararacussu and Crotalus durissus terrificus. In addition,
hydroalcoholic extracts from plants containing tannic acid (Plathymenia reticulata Benth) and
coumarin (Mikania laevigata Schultz Bip. ex Baker) were assayed against B. jararacussu
venom.
Male Swiss white mice (26-32 g) were supplied by Anilab - Animais de Laboratório
(Paulínia, São Paulo, Brazil). The animals were housed at 25 ± 3°C on a 12-h light/dark cycle
and had access to food and water ad libitum. This project (protocol number A078/CEP/2007)
was approved by the institutional Committee for Ethics in Research of Vale do Paraiba
University (UNIVAP), and the experiments were carried out according to the guidelines of
the Brazilian College for Animal Experimentation.
Venoms
Crude venoms were obtained from adults Bothrops jararacussu (Bjssu) and Crotalus
durissus terrificus (Cdt) snakes (Serpentário do Centro de Estudos da Natureza) and certified
by Prof. Dr. José Carlos Cogo, Vale do Paraíba University (UNIVAP, São José dos Campos,
SP, Brazil).
Phytochemicals
Tannic acid and coumarin were purchased from Sigma-Aldrich (USA) and used as
standard phytochemicals.
M. Laevigata Extract
The leaves of M. laevigata (1 kg) were harvested from plants at the University of
Sorocaba (UNISO) herbarium. A voucher specimen was deposited in the UNISO herbarium.
The powder (1 kg) obtained from M. laevigata leaves were percolated during 10 days in 50%
hydroalcoholic solution. After this period, the solution was dried at 40ºC by using forced air
circulation (dryer Marconi, São Paulo, Brazil) and crushed (10 Mesh, 1.70 mm) by using a
macro mill (type Wiley, MA 340 model, Marconi, São Paulo, Brazil). After this procedure,
252 Rafael da Silva Melo, Nicole Moreira Farrapo et al.
50% hydroalcoholic solution (Synth, São Paulo, Brazil) was added until complete extraction
(when the solution was incolor). Then, the extract was evaporated until dryness by using a
rotatory evaporator (Tecnal, São Paulo, Brazil) at 50 ºC. The resulting powder (131.68g) was
stored at room temperature and protected from light and humidity until the assays.
P. Reticulata Extract
The barks from P. reticulata were collected in October 2006 in Miracema city, Tocantins
State, Brazil. A specimen was deposited (protocol NRHTO 3327) at the herbarium of Federal
University of Tocantins (UFT). The barks were dehydrated in a stove at 37oC during 48
hours, powdered, ground in a mill, macerated with alcohol (70%) during 24 hours and
percolated in order to obtain a 20% (m/v) hydroalcoholic extract [27].
The proteins in the extract solutions were precipitated [14] with 1.0 mg/mL bovine serum
albumine (BSA, fraction V, Sigma) solution in 0.2 M acetate buffer (pH 4.9). After
centrifugation, the precipitate was dissolved in sodium dodecyl sulfate
(Sigma)/triethanolamine (Merck) solution and the tannins were complexed with FeCl3. The
supernatant of each extract (containing free tannins) was used for venom neutralizing assays,
and the coloring complex was spectrophotometrically read at 510 nm for tannins
determination [13, 14]. The tannin concentration in the samples was measured through a
standard curve obtained by a polynomial regression y=1.754x–0.1253 (R2=0.9971). Tannic
acid was used for the standard curve. All solutions were analyzed in triplicate.
Aliquots of the M. laevigata and P. reticulata hydroalcoholic extracts (MlHE and PrHE,
respectively) were spotted in thin-layer silica gel plates (0.3 mm thick, Merck, Germany) with
appropriate standards [15, 30]. The solvent system consisted of acetone:chloroform:formic
acid (10:75:8, v/v). The phytochemical groups used as standards (1% methanol - m/v, Sigma-
Aldrich, USA) were coumarin and tannic acid. The separated spots were visualized with
NP/PEG as following: 5% (v/v) ethanolic NP (diphenylboric acid 2-aminoethyl ester, Sigma,
Switzerland) followed by 5% (v/v) ethanolic PEG 4000 (polyethylene glycol 4000, Synth,
Brazil), being visualized under U.V. light at 360 nm. The retention factor (Rf) of each
standard was compared with those spots exhibited by both extracts obtained from M.
laevigata and P. reticulata.
The phrenic nerve-diaphragm muscle [3] was obtained from mice previously anesthetized
with halotane and sacrificed by exsanguination. The diaphragm was removed and mounted
under a tension of 5 g in a 5 mL organ bath containing aerated Tyrode solution (control).
Antiophidian Mechanisms of Medicinal Plants 253
After equalization with 95% O2 and 5% CO2, the pH of this solution was 7.0. The
preparations were indirectly stimulated with supramaximal stimuli (4 x threshold, 0.06 Hz,
0.2 ms) delivered from a stimulator (model ESF-15D, Ribeirão Preto, Brazil) to the nerve by
bipolar electrodes. Isometric twitch tension was recorded with a force displacement
transducer (cat. 7003, Ugo Basile), coupled to a 2-Channel Recorder Gemini physiograph
device (cat. 7070, Ugo Basile) via a Basic Preamplifier (cat. 7080, Ugo Basile). PND
preparations were allowed to stabilize for at least 20 min before the addition of one of the
following solutions: Tyrode solution (control, n=7); phytochemicals (tannic acid or coumarin,
100 mg/mL, n=6 each); venoms (Bjssu 40 µg/mL, n=10; Cdt 15 µg/mL, n=7); P. reticulata
hydroalcoholic extract (PrHE, 0.06 mg/mL, n=8) or M. laevigata hydroalcoholic extract
(MlHE, 1 mg/mL, n=5). The neutralization assays were carried out after preincubating the
PND preparations with the Tyrode solution during 30 min. After preincubation, the following
substances were added to the bath: phytochemicals + Cdt or Bjssu (n=6, each); MlHE + Bjssu
ou Cdt venoms (n=19 and 6, respectively); PrHE + Bjssu or Cdt venoms (n=8 and 5,
respectively). In order to verify the influence of tannins on the venoms neutralization, extracts
free of tannins (-) were also assayed against the crude venoms.
Experimental Design
Figure 1. Experimental design showing the rationale steps of the study on the isolated preparation.
254 Rafael da Silva Melo, Nicole Moreira Farrapo et al.
Statistical Analysis
Each pharmacological protocol was repeated at least five times. The results were
expressed as the mean ± S.E.M. Student’s t-test was used for statistical comparison of the
data and the significance level was set at 5%.
100
80
40
20
0
0 20 40 60 80 100 120 Time (min)
100
80
Twitch tension (%)
60
40
20
0
0 20 40 60 80 100 120 Time (min)
Figure 3. Preincubation procedure. Crotalus durissus terrificus venom (Cdt) or Bothrops jararacussu
venom (Bjssu) were preincubated with tannic acid or coumarin, 30 min prior the pharmacological
assays. Note that tannic acid causes a visible protein precipitation, but not coumarin.
PrHE (1) showed two different substances with retention factor (Rf) of 1.8 cm and 5.2
cm. Tannic acid (2) showed substances with Rf of 1.9 cm and 4.5 cm. MlHE (6) showed four
substances with Rf 4.3 cm, 5.5 cm, 8.5 cm and 9.0 cm. Under the solvent and revelator
systems used, commercial coumarin was not visualized (5). When tannins were complexed
with bovine serum albumin (BSA), no substance was visualized in 3, 4 and 7, respectively
free of tannins (-), PrHE (-) and MlHE (-).
The pharmacological results using extracts free of tannins (-) are showed in Figure 6.
Note that only MlHE (-) partially protected against the paralysis of Bjssu venom (Figure 6A),
but not against Cdt venom (Figure 6B). It is known that in vivo Cdt venom triggers different
mechanism of action than Bjssu. The neurotoxicity induced by the Crotalus genus is
attributed to crotoxin, the main toxin from this venom [2, 7, 31], whereas the Bothrops genus
is mainly myotoxic [22], due its main toxin, bothropstoxin-I [16]. Maybe plants having
coumarin could inhibit phospholipases with no catalytic activity (Lys49PLA2), as those found
in Bjssu venom, but they are not able to avoid the action of Asp49PLA2 triggered by Cdt
venom. Similar understanding was related by Cavalcante et al. [6] regarding to the ability of
aqueous extract of Casearia sylvestris against snake venoms phospholipase A2 toxins.
Antiophidian Mechanisms of Medicinal Plants 257
100
80
Twitch tension (%)
60
40
20
0
0 20 40 60 80 100 120 Time (min)
100
80
Twitch tension (%)
60
40
20
0
0 20 40 60 80 100 120 Time (min)
The isolation of substances from potential medicinal plants, even at high costs, to try to
keep their pharmacological effect at the end of the process is a modern tendency. We
presented a rationale experimental design, which tests probably phytochemicals before their
commercial availability. Our results showed that isolated compounds not always preserve the
same pharmacological efficacy as that seen with total extract.The results allowed proposing
the following different mechanisms by which plants can neutralize crude venoms: 1)
complexing proteins in vitro (as verified in tannic acid) or 2) mechanistically by a
phytocomplex formation.
We also observed the interference of phytochemicals free of tannins (-) such as the ones
showed in the chromatoplaque (Figure 5).
Figure 5. Thin layer chromatography. Extracts + phytochemicals with ou without tannins (-).
Chromatographic profile of (1) or M. laevigata (6) hydroalcoholic extracts. Phytochemicals are showed
in 2 (tannic acid) and 5 (coumarin). The withdrawal of tannins (-) from the tannic acid and P. reticulata
and M. laevigata hydroalcoholic extracts and are showed in 3, 4 and 7, respectively.
The isolation of substances from potential medicinal plants, even at high costs, to try to
keep their pharmacological effect at the end of the process is a modern tendency. We
presented a rationale experimental design, which tests probably phytochemicals before their
commercial availability. Our results showed that isolated compounds not always preserve the
same pharmacological efficacy as that seen with total extract.The results allowed proposing
the following different mechanisms by which plants can neutralize crude venoms: 1)
complexing proteins in vitro (as verified in tannic acid) or 2) mechanistically by a
phytocomplex formation.
Antiophidian Mechanisms of Medicinal Plants 259
140
120
100
Twitch tension (%)
80
60
40
20
0
0 20 40 60 80 100 120 Time (min)
140
120
100
Twitch tension (%)
80
60
40
20
0
0 20 40 60 80 100 120 Time (min)
ACKNOWLEDGMENTS
This work was supported by a research grant from Fundação de Amparo à Pesquisa do
Estado de São Paulo (Proc. FAPESP 04/09705-8) and PROBIC/UNISO. R.M.S. was granted
a scholarship (I.C.) from PIBIC/CNPq.
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In: Flavonoids: Biosynthesis, Biological Effects… ISBN: 978-1-60741-622-7
Editor: Raymond B. Keller © 2009 Nova Science Publishers, Inc.
Chapter 9
Kenichi Yoshida∗
Department of Life Sciences, Meiji University, 1-1-1 Higashimita,
Tama-ku, Kawasaki, Kanagawa 214-8571, Japan
ABSTRACT
There are serious concerns about the increasing global cancer incidence. As currently
used chemotherapeutics agents often show severe toxicity in normal cells, anti-
carcinogenic compounds included in the dietary intakes of natural foods are expected to
be applicable to a novel approach to preventing certain types of cancer without side
effects. Polyphenolic compounds, such as flavonoids, are ubiquitous in plants and are
presently considered to be the most promising in terms of having anti-carcinogenic
properties probably due to their antioxidant effect. To gain further insights into how
flavonoids exert anti-carcinogenic actions on cancer cells at the molecular level, many
intensive investigations have been performed. Currently, the common signaling pathways
elicited by flavonoids are recognized as tumor suppressor p53 and survival factor AKT.
These factors are potential effectors of flavonoid-induced apoptosis via activation of Bax
and caspase family genes. The present chapter emphasizes pivotal molecular mechanisms
underlying flavonoid-induced apoptosis in human cancer cells. In particular, this chapter
focuses on representative flavonoids such as soy isoflavone, green tea catechin, quercetin,
and anthocyanin.
INTRODUCTION
A major part of cancer incidence is thought to be related to life style factors such as
dietary intake tendencies. Considerable attention has been paid to bioactive polyphenols,
especially flavonoids, in dietary intake of many fruits and vegetables for the sake of cancer
prevention, because human population or epidemiologic studies have suggested that flavonoid
∗
Tel. and Fax.: +81-44-934-7107, e-mail: yoshida@isc.meiji.ac.jp
264 Kenichi Yoshida
intake may reduce the risk of specific types of cancer [1-3]. Flavonoids have been extensively
investigated in terms of how they act on various signal transduction pathways and their
influences on the processes of cell fate determination [4, 5]. Induction of apoptosis in cancer
cells by flavonoids is one of the most promising lines of evidence. Apoptosis induced by
flavonoids usually involves modulating p53, nuclear factor-kappaB (NF-kappaB), activator
protein-1 (AP-1), or mitogen-activated protein kinases (MAPK) [6-8]. Natural products are
being investigated that can antagonize the anti-apoptotic effects of Bcl-2 family proteins such
as Bcl-xL and Bcl-2 [9].
The changes which transform normal cells into cancer cells have been characterized as
successive molecular events including activation of oncogenes and inactivation of
antioncogenes, and all of these genes are known to be essential components of cellular signal
transduction pathways and effectors of the cell cycle and apoptosis control. Basically, loss of
anti-oncogenes results in the fragility of chromosomes and oncogenic activation renders cells
more resistant to apoptosis and metastatic phenotypes. Needless to say, in considering cancer
cell control via inhibition of metastasis and angiogenesis, inducing immune response and
inflammatory cascade specific for cancer cells, and modulation of drug resistance are all
promising approaches. In this chapter, we summarize recent progress in molecular studies of
major flavonoids acting on apoptotic cancer cells.
and Bax during EGCG-induced apoptosis has also been shown in human breast cancer cells
MDA-MB-468 [31]. Using different prostate cancer cell lines, p53 downstream targets p21
and Bax have been shown to be essential for EGCG-induced apoptosis [32]. There is also a
controversial report about p53, but this is not the case for p21, being dispensable for apoptosis
in human prostate carcinoma cells [33]. Other than p53, EGCG has been shown to affect
many cell cycle regulators such as the cyclin-dependent kinase (CDK) inhibitor and the
retinoblastoma gene product (pRb)-E2F in different cancer cells [34-37]. Recent findings on
EGCG-induced apoptosis in cancer cells favor an essential role of p53 in EGCG-induced
apoptosis. Additionally, survival factor AKT tends to be suppressed by EGCG in many
cancer cells. These two pathways appear to synergistically induce cell cycle deregulation and
alteration of Bcl-2/Bax balance, thereby leading to the induction of apoptosis in cancer cells.
2. SOY ISOFLAVONES
Genistein, which is structurally related to 17beta-estradiol, is one of the predominant
soybean isoflavones. Genistein has been shown to inhibit protein tyrosine kinase activity,
especially those of epidermal growth factor receptor and Src tyrosine kinase [38, 39], and is
implicated in protection against different cancers [40]. By regulating a wide array of genes,
genistein regulates the cell cycle and apoptosis. The common signaling pathways inhibited by
genistein for the prevention of cancer have been extensively examined and are known as the
NF-kappaB and AKT signaling pathways [41-45]. For example, apoptosis in many cancer
cells including the breast cancer cell line MDA-MB-231, prostate cancer cell line PC3, and
head and neck cancer cells treated with genistein has been shown to be executed partly
through the down-regulation of NF-kappaB and AKT pathways [46-48].
In contrast to apoptosis induced by EGCG in cancer cells, accumulating evidence
suggests that p53 does not appear to play a significant role during apoptosis in cancer cells.
Genistein induced apoptosis in a variety of human cancer cells regardless of p53 status [49].
A p53-independent pathway of genistein-induced apoptosis in non-small cell lung cancer
cells H460 has also been reported [50]. Although Puma, a p53-induced apoptosis regulator,
has been identified as an up-regulated gene in genistein-induced apoptosis of A549 cells, its
down-regulation had no effects on genistein-induced apoptosis [51]. p21, p53-target gene, has
been shown to be an important candidate molecule in determining the sensitivity of normal
and malignant breast epithelial cells to genistein and also in genistein-induced apoptosis in
prostate adenocarcinoma and non-small lung cancer cell line H460 [52-55]. Notably, p21
induction by genistein was reported to function in a p53-independent manner in human breast
carcinoma cells and prostate carcinoma cells [56, 57]. Genistein-induced apoptosis in primary
gastric cancer cells is partly explained by down-regulation of Bcl-2 and up-regulation of Bax
[58]. Along with p21, Bax was detected in genistein-induced apoptosis of the breast cancer
cell line MDA-MB-231 through a p53-independent pathway [59]. Taken together, these
observations indicate that genistein induces apoptosis in variety of cancer cells largely via
inhibition of AKT and NF-kappaB pathways, and that p53 involvement in genistein-induced
apoptosis may well be minor while p21 and Bax play certain roles in genistein-induced
apoptosis.
266 Kenichi Yoshida
3. QUERCETIN
Quercetin is a ubiquitous bioactive plant flavonoid found in onions, grapes, green
vegetables, etc., which has been shown to inhibit the proliferation of a variety of human
cancer cells. Quercetin is thought to induce apoptosis possibly through regulating MAPK
such as by suppressing extracellular signal-regulated kinase (ERK) and c-Jun N-terminal
kinase (JNK) phosphorylation [60]. Tumor necrosis factor (TNF) alpha-induced apoptosis in
osteoblastic cells, MC3T3-E1, was shown to be promoted by quercetin when JNK and AP-1
were activated [61]. TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis and
the resultant caspase activation was also promoted by quercetin in human prostate cancer
cells with accompanying suppression of AKT phosphorylation [62]. In human prostate cancer
cells, LNCaP, quercetin-induced apoptosis was accompanied by a decrease in the inhibitory
AKT Ser473 phosphorylation and its downstream Bad Ser136 phosphorylation. These
processes can promote dissociation of Bax from Bcl-xL and then Bax translocation to the
mitochondrial membrane, and induce activation of caspase family genes [63]. In a human
hepatoma cell line HepG2, quercetin induced apoptosis possibly by direct activation of the
caspase cascade and by inhibiting survival signaling such as AKT [64]. These results indicate
that quercetin-directed suppression of AKT phosphorylation could be the major molecular
axis for quercetin-induced apoptosis in cancer cells.
p53 has been thought to have an important role in apoptosis in quercetin-treated cells
[65]; however, there is a discrepancy regarding the p53 requirement in quercetin-induced
apoptosis. Regardless of p53 status, quercetin was able to induce Bad and caspase family
genes and this resulted in apoptosis of nasopharyngeal carcinoma cell lines [66]. Quercetin
induced apoptosis in the human prostate cancer cell line PC-3 with increased levels of insulin-
like growth factor-binding protein-3 (IGFBP-3), Bax, and p21 protein and with decreased
levels of Bcl-xL and Bcl-2 proteins, suggesting that quercetin-induced apoptosis may occur in
a p53-independent manner because PC-3 lack p53 [67, 68]. In contrast to the above reports,
quercetin has been shown to induce cell cycle arrest and apoptosis in human hepatoma cells,
HepG2, in a p53-dependent manner, and this resulted in an increased ratio of Bax/Bcl-2 [69].
Moreover, p53-dependent up-regulation of p21 has been shown to attenuate apoptosis in
quercetin-treated A549 and H1299 lung carcinoma cells [70]. An important role of p21 in
quercetin-induced apoptosis of MCF-7 human breast cancer cells has been reported [71].
Taken together, these findings indicate that quercetin-induced apoptosis in cancer cells
requires both inactivation of survival factor AKT and modulation of the expression of the
Bcl-2 family of proteins, especially for Bax activation. Recent findings suggest that quercetin-
induced apoptosis does not apparently requir p53, though more extensive studies are needed.
Results obtained to date indicate that quercetin can be applied to a wide variety of cancer cells
that lack p53.
4. ANTHOCYANINS
Anthocyanins, reddish pigments, are abundant in many fruits and vegetables such as
blueberries and red cabbage. As is well known for other flavonoids, anthocyanins have the
ability to act as antioxidants and are expected to be of potential clinical relevance as
Molecular Targets of Flavonoids during Apoptosis in Cancer Cells 267
evidenced by animal models [72]. Moreover, berry phenolics, in which the major components
are anthocyanins, have been known to show anti-carcinogenic properties mainly through the
induction of apoptosis in multiple types of cancer cells [73-76]. Among anthocyanins,
malvidin, has been shown to be the most potent apoptosis inducer by modulating specifically
MAPK in the human gastric adenocarcinoma cell line AGS [77]. Anthocyanins, derived from
potato, induced apoptosis in the prostate cancer cell line LNCaP and PC-3 accompanied by
MAPK and JNK activation, but caspase-dependent apoptosis was observed only in LNCaP
cells [78]. Hibiscus anthocyanins induced apoptosis in human promyelocytic leukemia cells,
HL-60, and this was critically regulated specifically by p38 kinase in MAPK and PI3-K [79].
In the human hepatoma cell line HepG2, delphinidin, components of anthocyanins induced
apoptosis with increased JNK phosphorylation and Bax and decreased Bcl-2 protein [80].
Molecular targets of anthocyanins likely include two regulatory axes: 1) MAPK activation
followed by JUN phosphorylation and 2) p53 activation followed by Bax protein activation.
These two pathways are known to contribute equally to apoptosis in normal cells [81]. The
contribution of p53 status in anthocyanin-induced apoptosis should be further verified by
studying a series of cancer cells.
CONCLUSION
Flavonoids are rich sources of potentially useful medical compounds, because they have
been well characterized as reducing cancer risk. Indeed, flavonoids are known to exhibit
antioxidant and anti-inflammatory actions as well as anti-carcinogenic effects on many types
of cancer cells. Cell culture-based experiments have revealed numerous candidate molecules
for development of flavonoids for drug targeting; however, the results of assays with very
high concentrations of flavonoids do not necessarily support the adaptablility of these
compounds to clinical treatments. The results cell culture-based experiments should be
verified and confirmed first using animal models and then in epidemiological studies.
Molecular mechanisms of flavonoid actions on cellular targets are not fully characterized and
many features remain to be elucidated. For example, the synergistic actions of flavonoids
should be carefully investigated, because we usually ingest multiple flavonoids
simultaneously from dietary sources, such as fruits, and beverages. Moreover, combination
effects between flavonoids and cancer preventive chemotherapeutic agents are a promising
approach to reducing side effects. Exploring the actual molecular targets of flavonoids would
presumably be a very promising road to the control cancer cells.
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272 Kenichi Yoshida
Chapter 10
ABSTRACT
Flavan-3-ols with the most well known members being catechin and epicatechin are
a group of phenolic compounds widely distributed in nature. The oligomers and polymers
of flavan-3-ols are known as condensed tannins which used to be considered as anti-
nutritional components. In recent years, more and more evidences proved that these
compounds were beneficial to human health as nutrition and lifestyle have fundamentally
changed in modern society. These phenolic compounds showed great potential for the
treatment of lifestyle related diseases, such as type 2 diabetes, obesity, and metabolic
syndrome. They were also reported to have effects on slowing down the aging progress
as well as on prevention of Alzheimer’s disease, cardiovascular disease, and cancer. This
chapter describes the structures, chemical properties, isolation and identification methods,
bioactivity and distribution of flavan-3-ol monomers and condensed tannins in dietary
sources and medicinal plants. Case studies such as the chemical and biological
investigations of tannins in the stems of Cynomorium songaricum (a well known tonic in
China) and in other plants are provided.
INTRODUCTION
Flavan-3-ols with the most well known members being catechin and epicatechin are a
group of phenolic compounds widely distributed in nature. The oligomers and polymers are
known as proanthocyanidins or in another word, condensed tannins, characterized by their
∗
Corresponding authors: Phone: (81)-76-4347633. Fax: (81)-76-4345060. Email: saibo421@inm.u-toyama.ac.jp
(M. Hattori); ma@inm.u-toyama.ac.jp or mchaomei@hotmail.com (CM. Ma).
274 Chaomei Ma and Masao Hattori
typical astringency taste. This sensory property causes the dry and puchery feeling in the
mouth following the consumption of tea, red wine and some unripened fruits
(http://en.wikipedia.org/wiki/Tannin). Tannins used to be considered as anti-nutritional
components due to their ability to bind with proteins. However, as nutrition and lifestyle have
fundamentally changed in modern society, more and more evidences proved that these
compounds have beneficial effects to human health (Ren and Chen, 2007; Shahidi, 1997). As
polyphenol compounds with potent anti-oxidative activity, they showed effects on slowing
down the aging progress as well as on prevention of Alzheimer’s disease, cardiovascular
disease, and cancer. These phenolic compounds also showed potential for the treatment of
lifestyle-related diseases, such as type 2 diabetes, obesity, and metabolic syndrome. There is
an excellent review well discussed the bioactivities of tannins from edible sources, especially
their antioxidant and radical scavenging activities, their prevention effects of cancer and
cardiovascular diseases, inhibition of LDL oxidation and platelet aggregation effects (Santos-
Buelga and Scalbert, 2000). The present chapter describes more about the chemical aspect of
these compounds, including the structures, chemical properties, isolation and identification
methods of flavan-3-ol monomers and condensed tannins in dietary sources. Their updated
bioactivities will also be discussed. Case studies such as the chemistry and bioactivity of
tannins in teas, peanut skins, grapes, wine and in the stems of Cynomorium songaricum (a
well known tonic in China) are presented.
OH OH
3' OH OH OH
2' 4'
1
8 HO O HO O
HO 7 O 2
5'
1'
6'
OH
3
6
OH OH OH
5 4
OH OH
OH afzelechin
catechin gallocatechin
OH OH
OH OH OH
HO O HO O HO O
OH
OH OH OH
OH OH OH
epicatechin epigallocatechin epiafzelechin
Flavan-3-ol monomers are the basic component units of oligomers and polymers (often
called condensed tannin). Epicatechin/catechin are the most frequently used extending flavan-
3-ol units in tannin structures. Sometimes epicallocatechin/gallocatechin and
epiafzelechin/afzelechin are also found as the extending flavan-3-ol units.
Dimers of flavan-3-ols linked through one C-C bond are named as procyanidin Bs. There
are many isomers of procyanidin Bs differed by the nature of their component monomer
units, order of the monomers, configurations of the linkage bonds and the linkage positions.
The most common linkages are C4→C8 (such as procyanidins B1-B4, figure 2) or C4→C6
(such as procyanidins B5 and B6, figure 2). Most prodelphinidins are flavan 3-ol dimers using
one or two gallocatechin/epigallocatechin as their monomer unit(s), such as prodephinidin B1.
A-type procyanidins are flavan-3-ol dimers linked through one ether bond in addition to a C-
C bond. The most common procyanidin As are procyanidins A2 and A1 (Figure 3).
Procyanidin Cs are flavan-3-ol trimers (Figure 4). The following figures show the structures
of some representative flavan-3-ol oligomers. Please note that though in most cases
procyanidin Bx is the same compound of proanthocyanidin Bx (where x is a certain number),
sometimes they are different compounds. For example, procyanidin B6 and proanthocyanidin
B6 are different, the former being a catechin (4→6) catechin dimer while the latter containing
a galloyl group conjugated with an epicatechin dimer (Scifinder). Most flavan 3-ols link with
C4→C8/C4→C6 bonds in larger oligomers and polymers (Figure 5). Some times procyanidin
A units can also be found in the structures of oligomers and polymers of condensed tannins.
OH OH
OH OH
O HO O
HO
OH OH
OH OH OH
OH
OH OH
O HO O
HO
OH OH
OH OH
Procyanidin B1 Procyanidin B2
Epicatechin-(4β-8)-catechin Epicatechin-(4β-8)-epicatechin
Proanthocyanidin B1 Proanthocyanidin B2
Procyanidol B1 Procyanidol B2
Figure 2. (Continued).
276 Chaomei Ma and Masao Hattori
OH OH
OH OH
HO O HO O
OH OH
OH OH OH OH
OH OH
HO O HO O
OH OH
OH OH
Procyanidin B3 Procyanidin B4
Catechin-(4α-8)-catechin Catechin-(4α-8)-epicatechin
Proanthocyanidin B3 (-)-Procyanidin B4
Procyanidol B3 Procyanidol B4
HO
HO
HO
HO OH HO
OH HO
OH
OH O O
O O
OH
OH OHHO
HO
OH OH
OH HO
HO
Procyanidin B5 Procyanidin B6
Epicatechin-(4β-6)-epicatechin Catechin-(4α-6)-catechin
Proanthocyanidin B5 Procyanidol B6
Procyanidol B5
OH
OH HO
HO O HO
O OH HO
OH O OH OH
HO O O
OH
OH
O OH OH
OHHO
OH
OH
HO HO
OH
Procyanidin B7
Proanthocyanidin B6 Epicatechin-(4β-6)-catechin
Proanthocyanidin B7
Flavan-3-ol Monomers and Condensed Tannins in Dietary and Medicinal Plants 277
OH
OH
HO
HO O
OH
HO OH
OH HO OH
OH OH
OH
O O
HO O
OH OH
OH HO
OH
OH
HO OH
Procyanidin B8 Prodelphinidin B1
Catechin-(4α-6)-epicatechin Epigallocatechin-(4β-8)-gallocatechin
Procyanidol B8
OH
OH OH
OH
HO O
HO O
O
OH O
OH
HO
HO
O OH
HO O OH
HO
OH
HO OH
HO
Proanthocyanidin A1 Proanthocyanidin A2
Epicatechin-(2β-O-7, 4β-8)-catechin Epicatechin-(2β-O-7, 4β-8)-epicatechin
OH
OH
HO O
OH
OH OH
OH
HO O
OH
OH OH
HO
HO O
OH
OH
Procyanidin C1
OH
HO
HO
R
n R=H or OH
R OH
Astringency and bitterness are the characteristic tastes of tannins. A study has showed
that these sensory properties are related to the structures especially for flavan-3-ol monomers,
dimers and trimers. The monomers were significantly higher in bitterness than the dimers,
which were significantly higher than the trimers. Astringency of the monomers was lower
than the dimers or trimers. The linkages between the monomeric units also influence both of
the sensory properties. Catechin-(4→6)-catechin was more bitter than catechin-(4→8)-
catechin/epicatechin. Astringency was affected by both the linkage and the identity of the
monomeric units. Catechin-(4→8)-catechin had lower astringency than either catechin-
(4→6)-catechin or catechin-(4→8)-epicatechin (Peleg et al., 1999).
Chemical Properties
bridge between the 3-O group on ring C and C6′ on ring B (Figure 6). The planer catechin
derivatives showed potent anti-oxidative activity and strong α-glucosidase inhibitory activity,
suggesting the potential of using these planer catechin derivatives as lead compounds for the
development of anti-diabetic agents [Hakamata et al., 2006; Fukuhara et al., 2002]. The
highly nucleophilic C-8 position makes it possible to react with some electrophilic reagents to
yield the C-8 substituted catechin derivatives (Figure 6). These compounds were synthesized
upon protection of the phenolic OH groups with benzyl groups and de-protection with
hydrogenation after the reaction finished (Nour-Eddine et al., 2007).
OH OH OH
OH OH R OH
HO O HO O HO O
OH R OH
O
OH S R OH
OH
R=(CH2)nCH3, (n=0-5) R=CHO/CH2OH/CH3/Br/CHOHCF3/COCF3
H2N COOH C-8 substitited catechin derivatives
Planer catechin derivatives
Catechin-cysteine conjugate
many new flavan 3-ol oligomers from various plants. Their published papers displayed
detailed spectral data of the condensed tannins. These spectral data can be used to assistant
structure determination of pure tannin compounds isolated from other plant sources (Hwang
et al., 1989; Morimoto et al., 1987; Morimoto et al., 1988; Kashiwada et al., 1986; Hsu et al.,
1985).
degradation products could qualitatively and quantitatively determine the extender units of
condensed tannins (Matthews et al., 1997; Santos-Buelga and Scalbert 2000).
ESI and API-MS are also used to study flavan-3-ol oligomers. TOFMS can be used to
analyze flavan-3-ol oligomers/polymers with higher molecular weights. The MS method can
provide information about the polymerization degree and the type of the component monomer
units. However, it is hard to determine the exact structures of these condensed tannins only by
MS and accurate quantification of these compounds by MS is difficult to achieve, especially
for larger oligomers or polymers.
Peanut skins are used to treat chronic haemorrhage and bronchitis in Chinese traditional
medicine. The skins contain both B-type and A-type proanthocyanidins. The B-type dimers
include B2, B3 and B4.
The A-type dimers, with one C-C bond and one ether bond linkages, include
proanthocyanidin A1 (p1), proanthocyanidine A2 (p2), epicatechin-(2β-O-7, 4β-8)-ent-
epicatechin (p3), epicatechin-(2β-O-7, 4β-6)-catechin (p4), epicatechin-(2β-O-7, 4β-6)-ent-
catechin (p5) and epicatechin-(2β-O-7, 4β-6)-ent-epicatechin (p6). Peanut skins also contain
some oligomers having A-type proanthocyanidin unit, such as p7 and p8 (Figure 7). These
compounds may be responsible for the bitter taste of the peanut skins. Like other phenolic
compounds, these compounds showed free radical-scavenging effects, which could protect
the fatty acids in the peanut seeds from oxidation (Lou et al., 2004; Lou et al.,1999).
Tea Polyphenols
Teas contain large quantity of polyphenols with gallocatechins and their gallate esters
being among the main constituents. During fermentation process, the colorless (gallo)
catechins in fresh tea leaves are oxidized to theaflavins and thearubigins in the presence of tea
polyphenol oxidase and peroxidase and under the influence of pH. Theaflavins display a
bright orange-red color and thearubigins display a red-brown or dark-brown color in solution.
The chemical structures of theaflavins contain a substituted benzotropolone moiety formed
from two flavan 3-ol units (Figure 8). Thearubigins are heterogeneous polymers whose
chemical structures have not yet been completely characterized (Davis et al., 1997; Takino et
al., 1965).
Tea flavan 3-ols exhibited a wide range of health beneficial effects. Professor Yokozawa
and her group in University of Toyama, Japan, have worked on pharmacological evaluation
of green teas and the components for decades. Their research group proved that the flavan-3-
ols had effects on scavenging of nitric oxide and superoxide and that gallic acid conjugates of
flavan-3-ols were more active than the un-conjugated ones. (-)-Epigallocatechin 3-O-gallate,
(-)-gallocatechin 3-O-gallate and (-)-epicatechin 3-O-gallate exhibited higher scavenging
282 Chaomei Ma and Masao Hattori
activity on both nitric oxide and superoxide than (-)-epigallocatechin, (+)-gallocatechin, (-)-
epicatechin and (+)-catechin did (Nakagawa and Yokozawa, 2002). These compounds
OH OH
OH
OH OH
OH
HO O HO O
HO O
O
O O
OH OH
OH
OH OH
OH
O OH
O OH O OH
HO OH
HO OH HO OH
OH p3
OH p1 OH p2
OH OH
OH OH
OH
OH
HO O HO O
HO O
O O
O OH OH
OH OH
OH
OH
HO O HO O
HO O
OH OH OH OH
OH OH
OH p6 OH
p4 OH p5
OH
OH OH
OH
OH
HO O OH
HO O O OH OH
HO O OH OH
O
OH OH
OH
O OH OH
OH OH O
HO O OH
OH O
HO OH
HO OH
OH OH OH HO
OH
p7 OH p8
Figure 7. Structures of some flavan-3-ol dimers and trimers isolated from peanut skins.
OH OH
OH
OH OH
OH
HO O HO O
OHOH OH HO O
OH
O OH O OH OH
OH OH
O O OH
OH OH
Epigallocatechin 3-O-gallate Epicatechin 3-O-gallate Epigallocatechin
OR2 OH
HO OH
R2O
O
O
OH HO
OH O OH
OH
HO O HO
OH OH
OH
OR1 HO O
OH
OH
Theaflavin R1=R2=H OR1
Theaflavin gallate R1=H, R2=galloyl or R1=galloy, R2=H OH
Theaflavin digallate R1=R2=galloyl Theasinensins R1,R2=H or galloyl
Flavan-3-ols in Wine
Phenolic compounds, mainly flavan-3-ols and anthocyanins play important roles in the
quality of wines. Their structures and amounts affect the color and sensory property such as
astringency of the wines. The type and quantity of flavan-3-ols in wines depend not only on
the kinds of the original fruits but also on the manufacture method, storage time and storage
temperature. Due to their high chemical reactivity, the phenolic compounds polymerize and
condense with other compounds in the wine solution to produce some special compounds for
individual wines. The condensation causes a change of color from light to dark during the
storage and aging process. The reaction of catechin condensed with glyoxylic acid, a
compound may be derived from tartaric acid, is such an example (Figure 9). This reaction is
generally observed during the aging of wines and other grape-derived foods (Es-Safi et al.,
2000.)
Flavan-3-ol related compounds, anthocyanins (Figure 10), can also undergo similar
condensation, such as with pyruvic acid, an end-product of the glycolysis cycle during
fermentation, to form stable red pigments as shown in Figure 11. Other yeast metabolites
other than pyruvic acid were also found to react with grape anthocyanins, suggesting that this
type of condensation may be an important route of conversion grape anthocyanins into stable
pigments during the maturation and ageing of wine, thus increase the color stability of aged
wines (Fulcrand et al.,1998).
284 Chaomei Ma and Masao Hattori
O
OH OH HO
OH HO
HO O O O OH
O OH OH
CHO COOH HO C C OH
OH CH COOH OH O
HO OH HO O
OH OH HO O
OH
OH +
OH
OH HO O OH
O O
HO COOH OH
OH HO
OH OH
Figure 9. Proposed products for the condensation of flavan-3-ol with glyoxylic acid.
OCH3
OCH3 OCH3
OH
OH OH
H+ +
+ HO O
HO O HOOC HO O OCH3
OCH3 OCH3
OH
OGlc
OGlc OGlc
O
OH O
HOOC OH COOH
Grape Tannins
Recent years have witnessed increasing interest in using grape seed extracts as active
ingredients in health promotion products. Grape seed tannins are a complex mixture of
oligomers and polymers composed of catechin, epicatechin and epicatechin-3-gallate.
Oligomers are mainly B-type linked through the C4→C6 or C4→C8 bond (Nu´ n˜ez et al.,
2006). Epicatechin was the major component in the extended chain while catechin was more
abundant in terminal units than in extension units. The proportion of galloyllated units varied
Flavan-3-ol Monomers and Condensed Tannins in Dietary and Medicinal Plants 285
from 13% to 29% as the Mr, increased. Degrees of polymerization ranged from 2.3 to 15.1 or
from 2.4 to 16.7 as measured by two different methods (Prieur et al., 1994).
Grape skins also contain tannins with epicatechin representing 60% of the extension
units, whereas 67% of the terminal units consisting of catechin. The degree of polymerization
ranged from 3 to 80. The proportion of galloyllated units was 3% to 6% (Souquet et al.,
1996).
Tannins in grape seeds are shorter and have more epicatechin gallate as the monomer
units. Tannins in grapes skin are generally larger and comprise more epigallocatechin as the
monomer units (Herderich and Smith, 2008).
Cynomorium Tannins
Figure 12. C. songaricum growing in the half-deserted areas of Inner Mongolia, China.
By separating the tannin fractions on Sephadex LH-20 and MCI gel CHP20P columns,
both with H2O-MeOH as mobile phases, we isolated procyanidins B1 and B6, flavan 3-ol
trimers, tetramers, pentamers and a mixture of higher oligomers and polymers. The two
isomers (procyanidins B1 and B6) of dimeric flavan 3-ols were separated as pure compounds.
The trimers, tetramers and pentamers were obtained as mixtures of isomers containing
epicatechin/catechin as the flavan 3-ol unit as indicated by their API-MS spectra. The
286 Chaomei Ma and Masao Hattori
OH OH
OH
HO
HO O
O PhCH2SH/H+
OH
HO OH S
OH
n Benzylthioepicatechin
OH
The contents of the phenolic compounds play important role in the antioxidant activity
and other related bioactivities of fruits and vegetables. Cai et al. have studied a large number
of plants about the relationship between their antioxidant activity and the contents of phenolic
compounds. It was found that the antioxidant activity and the total phenolic contents for the
tested herbs (112 species) were positively and significantly linearly correlated
(methanolic/aqueous: R2 = 0.964/0.953). Plants with higher total phenolic contents showed
higher antioxidant capacity. The study showed that the contents of phenolic compounds in
common fruits and vegetables were in the order of followings (from high to low phenolic
contents): eggplant, spinach, Chinese lettuce, Washington red apple, broccoli, orange, Fuji
apple, tomato, spring onion, kiwifruit, carrot, garlic, cucumber and pear. Tannins are among
the main components of the phenolic compounds in these plants (Cai et al., 2004).
Mai et al. investigated the α-glucosidase inhibitory and antioxidant activities of
Vietnamese edible plants and discussed their relationships with the polyphenol contents. The
results showed that the extracts from plants used for making drinks showed the highest
Flavan-3-ol Monomers and Condensed Tannins in Dietary and Medicinal Plants 287
activities, followed by edible wild vegetables, herbs, and dark green vegetables. Positive
relationships among α-glucosidase inhibitory activities, antioxidant activities and polyphenol
contents of these plants were found. All the 5 plants (Camellia sinensis (Che Xanh),
Cleistocalyx operculatus (Voi), Psidium guajava (Oi), Nelumbo nucifera (Sen), Sophora
japonica (Hoe)) used for making drinks had high enzyme inhibitory activities and high
antioxidant activities (Mai et al., 2007).
CONCLUSION
Flavan 3-ols consumed in our daily foods and beverages play important roles in helping
us to keep healthy. As potent antioxidants, they have effects on slowing down the aging
progress as well as on prevention of Alzheimer’s disease, cardiovascular disease, and cancer.
These compounds also have inhibitory activity on α-glucosidase, suggesting they may have
effect to prevent some modern diseases, such as type 2 diabetes, obesity, and metabolic
syndrome.
Chemically, these compounds are unstable. Their C8 and C6 are highly nucleophilic,
while C4 is electrophilic. Due to these chemical properties, these compounds are easy to
polymerize and to condense with other compounds co-existing in the dietary materials.
Specific controls of these reactions can produce dietary materials with special color, sensory
property and biological activity.
Separation and identification work for the flavan 3-ol conjugates are considerably more
difficult than for other types of small natural products, and thus the identification and
biological activity of pure tannin compounds have not been fully investigated.
While the healthy beneficial effects of tannins are emphasized in this paper and in many
other articles in recent years, caution will also be taken not to consume too much tannin
mixtures as the non-specific interactions of tannin mixtures with proteins may cause some
adverse effects. However, the current situation is that people, especially those in developed
countries, do not take enough phenolic compounds, which lead to increasing cases of obesity
and other modern diseases, thus, higher intake of tannin-containing vegetables and fruits in
daily diets is recommended.
REFERENCES
Cai Y, Luo Q, Sun M, Corke H. 2004. Antioxidant activity and phenolic compounds of 112
traditional Chinese medicinal plants associated with anticancer. Life Sci 74: 2157-2184.
Davis AL, Lewis JR, Cai Y, Powell C, Davies AP, Wilkins JPG, Pudney P, Clifford MN.
1997. A polyphenolic pigment from black tea. Phytochemistry 46:1397-1402.
Es-Safi N-E, Le Guerneve C, Cheynier V, Moutounet M. 2000. New phenolic compounds
formed by evolution of (+)-catechin and glyoxylic acid in hydroalcoholic solution and
their implication in changes of grape-derived foods. J Agric Food Chem 48: 4233-4240.
Ezaki-Furuichi E, Nonaka G, Nishioka I, Hayashi K. 1986. Tannins and related compounds.
Part XLIII. Isolation and structures of procyanidins (condensed tannins) from
Rhaphiolepis umbellata. Agric Biol Chem 50: 2061-2067.
288 Chaomei Ma and Masao Hattori
Chapter 11
ABSTRACT
Accurate taxonomic groupings are important for many applications, especially for
medicinal plants. For example, structural analogues of the antitumour agent, paclitaxel,
found in common Taxus species have increased the availability of this life-saving
medicine without relying on the slow growing and comparatively uncommon, T.
brevifolia [1]. Flavonoids have a long history of use as chemotaxonomic markers and
have assisted in resolving many taxonomic disputes that have arisen as a result of
morphological classification. In recent times, there has been increased interest in using
molecular systematics and bioinformatics as alternatives to traditional chemotaxonomic
techniques, however the investigation of the types flavonoids present in plants is still a
useful technique to rapidly assess plant taxonomy.
Planchonia careya is a medicinal plant that contains a range of antibacterial
compounds. Species of this genus are morphologically related to Barringtonia and
Careya species and there have been several changes of nomenclature to reflect the
uncertainty of these relationships.
Our recent investigation of some of the comprising flavonoids from Planchonia
careya has revealed similar distinctive compounds to those found in Planchonia grandis
that are notably absent from Barringtonia and Careya taxa. Therefore the comparatively
simple analysis of the flavonoid component of plant extracts can confirm or contest
phenetic groupings to help resolve taxonomic discrepancies.
292 Jacqui M. McRae, Qi Yang, Russell J. Crawford et al.
1. INTRODUCTION
Current taxonomic classifications group species into genera or families based on
morphological similarities. Although this method has proven to be satisfactory in most cases,
multiple revisions in the classification of many species highlight the inadequacies of phenetic
taxonomy [2]. A more precise method of classification is often required to accurately group
species, and molecular systematics and chemical taxonomy (also referred to as
chemotaxonomy or biochemical systematics) have been used in these applications. Molecular
systematics, including bioinformatics and DNA barcoding, is used to detect the presence of
certain genetic markers in related plant species, while chemotaxonomy focuses on the
similarities of compounds produced when a particular gene is expressed in related taxa [3, 4].
The utilization of both taxonomic methods can therefore provide a great deal of data for
understanding the functions of secondary metabolites in plants [2, 5].
Secondary metabolites commonly have restricted distribution patterns in plants, with
many occurring only in particular families or within species of a particular genus [1].
Morphologically-related plants are known to produce similar secondary metabolites and this
observation led to the development of chemotaxonomy. This concept was in its infancy in the
1950s and grew in prominence over the following two decades [6, 7]. The advent of DNA
sequencing technology in the 1990s eclipsed the enthusiasm for chemotaxonomy for a time,
however it has since been shown that both techniques are required to provide adequate data
for the phenetic and classification of a plant species [5, 7].
Chemotaxonomy ideally relies on the systematic analysis of particular phytochemicals in
all known species of each family or genus. Specific types of compounds that are present in
members of a particular genus and absent from representatives of a related genus (referred to
as chemotaxonomic indicators or markers), can be used to prove if the morphological
grouping is accurate. These compounds can also assist in the classification of new species.
Chemotaxonomic markers must have particular characteristics to be of value in systematic
investigations, including chemical complexity and structural variability, stability, and
widespread distribution in plant species [6].
Alkaloids are a structurally diverse class of compounds and have therefore been used as
chemotaxonomic markers [8, 9]. However, this class is limited in this application since these
compounds occur in less than 20% of angiosperm families [10]. Flavonoids are less diverse
than the alkaloids, with over 8 150 known structures reported up to the year 2006 [2]
compared with the 12 000 known alkaloids, although they appear to be almost universally
distributed among plant families. This makes them more widespread than other classes of
secondary metabolites and therefore flavonoids have been used extensively as
chemotaxonomic markers [2, 11].
Distribution patterns of flavonoid glycosylation and acylation in plant species have
shown strong correlations with morphologically related species. Chemotaxonomic
investigations of flavonoid glycosides have demonstrated that complex glycosidic patterns
occur in many plant families and these usually correlate with morphological classifications
[12]. For example, investigations of the flavonol tri- and tetraglycosides of Styphnolobium
and Cladrastis genera indicated that the morphological sub-grouping of some species may be
∗
Email: epalombo@swin.edu.au
Chemotaxonomic Applications of Flavonoids 293
inaccurate [13]. Further, the closely related genera, Pergularia and Gomphocarpus
(Asclepiadaceae), were confirmed as being distinct based on the presence or absence of
certain quercetin O-diglycosides and acylated kaempferol glycosides [14].
Chemotaxonomy has also resolved some issues that had arisen due to the difficulty of
morphological classifications. The controversial distinction between the genera Malus (apple)
and Pyrus (pear) was resolved by the presence of dihydrochalcones in all known species of
Malus, and the absence of such compounds from Pyrus species [6]. Further, the recent
creation of a separate grouping for some Ateleia species (Leguminosae) has been supported
by the discovery of unusual flavonoid glycosides in only some of these species. This study
also confirmed the placement of the Ateleia genus in the subfamily Papilionoideae, based on
the glycosylation pattern of the comprising flavonols, thus validating the morphological
classification [15]. Chemotaxonomic investigations of the types of anthocyanins present in
certain species of Lecythidaceae and Myrtiflorae have also supported the morphological
division of these families [16].
These correlations validate the use of phytochemicals in plant taxonomy and also the use
of flavonoids as chemotaxonomic indicators. In addition, the ongoing discovery of novel
flavonoid glycosides is a testament to their structural diversity and may yet reveal many novel
compounds.
sample. Improvements in the sensitivity of the instrument (greater than 500 MHz) as well as
the development of multibond heteronuclear experiments have enabled the complete
characterization of complex compound structures with less than one milligram of sample
[24].
NMR technology is continually being improved and recent developments include the use
of cryoprobes, which greatly increase the signal to noise ratio and therefore reduce the
experiment time or the amount of sample [25]. The use of submicro-inverse-detection
gradient NMR probes and other inverse detection techniques has also led to increases in the
sensitivity of NMR spectroscopy enabling the elucidation of compounds of less than 0.05
µmol [26]. The use of NMR spectroscopy has enabled the full structural elucidation of many
thousands of novel flavonoid glycosides that may have otherwise remained only partially
characterized.
Figure 1. Comparison of the flowers from Planchonia careya and Barringtonia racemosa [39, 40].
Figure 2. Compounds isolated from Planchonia sp.: 1-3, Acylated kaempferol hexaglycosides isolated
from P. grandis; 4, Acylated kaempferol tetraglycoside isolated from P. careya.
Many of these species have experienced similar taxonomic contention to P. careya so the
exact number of species remains uncertain. Chemotaxonomy is therefore a more useful
method of clarifying the classification of Planchonia taxa as distinct from other species.
Crublet et al. [33] isolated three novel acylated kaempferol hexaglycosides from the leaves of
P. grandis (1-3, Figure 2), yet no similar compounds have been isolated from Careya or
Barringtonia species. The presence of related compounds in P. careya would indicate that
acylated flavonol polyglycosides were effective chemotaxonomic markers for the Planchonia
genus [27].
To assess this premise, a collection of fresh P. careya leaves were crushed and extracted
with water by immersion in the solvent. After a total of three 24 hour extractions, the
flavonoid component of the crude aqueous extract was concentrated on XAD-16 resin and
eluted with methanol. Separation was achieved with reversed phase C18 (100-200 mesh)
media followed by Medium Pressure C18 (15 μm) media and isolation with preparative
reversed phase HPLC (5 μm) gave the novel acylated flavonol tetraglycoside, kaempferol 3-
O-[α-rhamnopyranosyl(1→3)-(2-O-p-coumaroyl)]-β-glucopyranoside, 7-O-[α-
296 Jacqui M. McRae, Qi Yang, Russell J. Crawford et al.
CONCLUSION
The advent of molecular systematics techniques has led to a significant reduction in
chemotaxonomic investigations in recent years. However, the capacity of chemotaxonomy as
a comparatively simple and reliable method of plant classification ensures the continued
validity of this technique.
ACKNOWLEDGMENTS
The authors would like to thank the Sunshine Foundation for providing financial support
for this research and Andrew Ford of the CSIRO Tropical Rainforest Centre for collecting the
plant material. We also thank Dr. Noel Hart for his expertise in methods of compound
separation and Dr. Roger Mulder for assistance with the NMR elucidation.
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Chemotaxonomic Applications of Flavonoids 299
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In: Flavonoids: Biosynthesis, Biological Effects… ISBN: 978-1-60741-622-7
Editor: Raymond B. Keller © 2009 Nova Science Publishers, Inc.
Chapter 12
ABSTRACT
Flavonoids are one of the most diverse and widespread groups of natural products.
They ubiquitously occur in all parts of plants including the fruit, pollen, roots and
heartwood. Plant extracts rich in flavonoids such as: Ginkgo biloba extract, soy bean
extract and green tea extract are popular dietary supplements. Numerous physiological
activities have been attributed to them and their potential roles in the prevention of
hormone-dependent cancers have been investigated. Over 5000 different flavonoids have
been described to date and they are classified into at least 10 chemical groups. Flavonoid
compounds usually occur in plants as glycosides in which one or more of the phenolic
hydroxyl groups are combined with sugar residues.
Quality control of these plant-extract products is problematic due to the many
varying factors in herbal medicine. Unlike synthetic drugs (in which the concentration
and activity of a single known bio-molecule is monitored), there are many uncertainties
in terms of species variation, geographical source, cultivation, harvest, storage and
processing techniques which may lead to a product of different quality and efficacy. To
evaluate the quality of flavonoids contained within plant extracts it is therefore very
important to develop an analytical method which can monitor the quantity and variety of
flavonoids efficiently. Also the study of the absorption and retention of these compounds
within individuals is more problematic as more than one active compound is ingested
from the plant extracts.
The challenge presented by such extracts is therefore to determine the different
flavonoid species present in any given extract and also to determine any modification or
fortification of the extract. Furthermore, Administration, Distribution, Metabolism and
Excretion (ADME) studies require the quantitative analysis of multiple components
rather than a single drug compound. The increase in the number of new flavonoid reports
302 Shujing Ding and Ed Dudley
is due to two main factors: the advances in methods of separation and the rapid
development and application of modern mass spectrometry (MS). Mass spectrometry
proved to be the most effective technique in flavonoids research both in plant extract and
in biological samples. In this expert commentary we will review the methods available
for studying this wide-ranging class of compounds and also how modern techniques have
been applied in order to "mine" the data obtained for flavonoid specific information from
a complex metabolomic analysis.
4'
1 1' B
O O O
7 2
A C
6 3
4 OH
5
O O O
4
4' O
1 A 1
O 1'
4' 6' 6 2
B
1' 5 3
4 O
2'
O
O
O
O
O
O
O
O
Isoflavanone Biflavonyl
The flavonoids are classified into at least 10 chemical groups. Figure 1 lists common
subclasses of flavonoids and also demonstrates the chemical numbering system applied to
both the atoms and rings (A, B and C) when comparing the structures of different flavonoids.
Flavonoid compounds usually occur in plants as glycosides in which one or more of the
phenolic hydroxyl groups are combined with sugar residues, forming an acid-labile glycoside
O-C bond. There are also other forms of conjugated sugars (e.g. D-glucose, L-rhamnose)
which contribute to the complex and diverse nature of the individual molecules that have been
identified.
methodology for monitoring the validity of any new or existing “natural product” [9]. This
requirement was included in the European Economic Council guideline 75/318 “Quality of
Herb Drugs” [10]. Taking Gingko Biloba extracts as an example, the flavonoids content is an
important parameter in terms of quality control. The applied threshold for commercial
standardized extracts of Ginkgo is that flavonoids constitute no less than 24% of the material
and terpene lactones no less than 6% [11]. However in North America such extracts are
considered to be a “dietary supplement” rather than a medicinal product and hence they do
not attract the same level of scrutiny compared to Europe. The analysis of the levels of the
flavonoids (considered to be – at least one of many – bioactive agents present in herbal
extracts) is therefore useful in order to demonstrate that the individual taking the extract is
obtaining the expected benefits from these herbal remedies. Furthermore, such analyses are
also invaluable for the purpose of identifying any artificial fortification of extracts which are
claimed to be “pure”. Any such analysis can be used to study the entire flavonoid content of
an extract and quantitate the levels of “active” flavonoids in a given sample (similar to any
drug quality control assay). Alternatively "fingerprint" assays compare the entire profile of
these compounds in a given sample to that of a “pure”, unfortified extract in order to ensure
that the extract has the properties associated with the herb. Such “fingerprint” analyses are
however complicated due to the fact that the flavonoid composition of any herbal extract may
be altered due to natural environmental or conditions in which the herb is grown prior to
harvesting and so identifying an “ideal” flavonoid profile for comparison with commercial
herbal extracts can be problematic. Despite these potential complications, these two major
routes of bio-analysis of the flavonoid content of herbal extracts are commonly employed.
Fingerprint analysis allows the evaluation of the quality of plant extracts and is able to
provide comprehensive (including flavonoids and other components determination) and semi-
quantitative information for complicated plant extract samples. More recently bioinformatic
programs (e.g. fuzzy influence diagrams) have become available and may be applied in order
to minimise subjective judgments when studying such data [12]. This development is a very
important step when the active component is unknown in such complex biological extracts.
Fingerprint analysis of flavonoids can be achieved by using several chromatographic
techniques including thin-layer chromatography (TLC) [13], high-performance liquid
chromatography (HPLC) [14], gas chromatography (GC) [15] high-performance capillary
electrophoresis (HPCE) [16] and high-speed counter-current chromatography (HSCCC) [17].
More recently, GC [18] and HPLC [19] in combination with mass spectrometry (MS) have
become a popular alternative for fingerprint analysis as they have the advantage of providing
more information compared to the previously applied techniques. Traditional quantitation of
flavonoids has been achieved by spectrophotometry [20] and HPLC [21]. Both of these
techniques require an acid hydrolysis step of the plant extract in order to convert the
conjugated flavonoids into flavonoid aglycones. For spectrophotometry, rutin is normally
chosen as the reference standard and only total flavonoid can be quantified as “rutin
equivalent”. HPLC is able to quantify individual flavonoids provided that their aglycone
counterparts are available as standard reference compounds and flavonoids are quantified as
aglycone equivalents in such assays. A 2-4 hours soxhlet extraction is required to convert the
flavonoid glycosides to aglycones for the above two analytical methods. There are also
reports of the quantitation of flavonoids in herbal remedies using GC/MS [22], offering better
Bioanalysis of the Flavonoid Composition of Herbal Extracts… 305
450
400
Number publications
350
300
250 HPLC
200 MS
150
100
50
0
2003 2004 2005 2006 2007 2008
Year
*
Note 2008 data from 1st January until 31st October only.
Figure 2. Article title search results for flavonoid analysis by HPLC and MS between 2003 and 2008.
Capillary electrophoresis separation coupled with UV or ESI-MS detectors has also been
applied to the study of flavonoids, offering an interesting alternative to HPLC separation [23].
In addition, UPLC is regarded as one way to achieve faster, more efficient separations with
liquid chromatography by using smaller particle size stationary phase materials, and this
separation technique has also applied to flavonoid analysis [24]. However, as a separation
technique, HPLC is the more widely applicable and offers a larger range of choices (in terms
of solid phases and column sizes) and when coupled to mass spectrometry detection it can
provide structure-selective information concerning the analytes. This is very important in
matrices as complicated as plant extracts. Hence, whilst HPLC-UV detection analysis remains
well used for flavonoid analysis, mass spectrometry is becoming increasingly used also for
such studies as can be seen Figure 2. Reports of the application of HPLC/MS for flavonoid
analysis have more than doubled in the recent five years comparing to the previous five years.
In this review therefore, we propose to focus on the mass spectrometric properties of
flavonoids and mass spectrometric analysis of flavonoids in both plant extract and biological
samples.
glycoside groups attached in a different orientation will give only one signal, for example, 3-
O-[6-O- (α-L-rhamnosyl)-β-D-glucosyl] kaempferol and 3-O-[2-O-(β-D-glucosyl)-α-L-
rhamnosyl] kaempferol, both of which exist in Ginkgo biloba extracts [26].
400
350
Peak area/10E+5
300
250 [M+H]+
200 [M+Na]+
150 [M-H]-
100
50
0
IR KF QD QG QH RH
Figure 3. Comparison of ion intensity in positive and negative mode for some flavonoid components in
Ginkgo biloba.
Figure 4. Mass spectrum of a direct infusion of a G. biloba extract on an accurate mass – mass
spectrometer. (provided by Dr C. Williams, EPSRC National Mass Spectrometry Service Centre,
Swansea University)
308 Shujing Ding and Ed Dudley
The m/z of both is 593Th as they exhibit identical empirical formulae, and hence these
compounds cannot be distinguished by this method. As a result it is impossible to specify the
exact nature of individual moieties as different arrangements of the sugars attached to the
agylcone would generate identical data.
A further complication during accurate mass analysis arises due to the possibility of ion
suppression. This occurs due to the fact that a specific amount of “charge” is available for the
ionisation of the sample and hence the compounds present in the mixture “compete” for this
charge. The presence of an easily ionised compound within the mixture may therefore limit
the ionisation of others and hence the absence (or reduction in intensity) of a flavonoid ion
may not represent an absence (or decrease in level) of the compound but may arise due to this
factor. From this spectrum it can be noticed that Ginkgo biloba terpene lactone signals
significantly suppressed the ionisation of flavonoids, despite the fact that the content of
flavonoids is much higher than terpene lactones in the extract.
In order to address both of these issues, the application of a separation technique –
preferably directly linked to the mass spectrometer – can be utilised. HPLC analysis is again
applied with reverse phase (C18) stationary phases being the most commonly used. Recent
developments in the miniaturisation of HPLC columns, and the systems used to run them,
have led to lower mobile phase flow rates being applied in HPLC-MS analyses. This
miniaturisation, due to the concentration sensitive nature of electrospray ionisation source of
the mass spectrometer, leads to a significant increase in sensitivity. The reduction of the
internal diameter of the HPLC from 4.6 mm to 0.3 mm (and reduction in required mobile
phase flow rate from 1mL/min to 4μL/min) has been shown to result in a 80-100 fold increase
in sensitivity of the HPLC-MS assay [27, 28]. The HPLC separation prior to analysis allows
isobaric flavonoids to be separated and hence individually analysed and also removes the
potential inaccuracy of the data obtained due to ion suppression effects. In order to further
alleviate the problem of isobaric flavonoids in the extract, the combination of the HPLC
separation of flavonoids with tandem mass spectrometry (MS/MS and MSn analysis) - in
which the ions are fragmented with an inert “collision” gas and the fragment ions determined
– allows an information rich analysis to be performed without bias towards any particular
flavonoid.
Bioanalysis of the Flavonoid Composition of Herbal Extracts… 309
MS/MS requires the isolation of the ion of interest - the precursor ion- which is then
fragmented to generate “product ions”. These product ions can themselves be individually
isolated and fragmented further in an MS3 experiment. This type of analysis can be done in a
“data dependent” fashion in which the mass spectrometer “decides” which ion to fragment
(based on ion abundance and how often the ion is detected) and which of its product ions to
fragment further. This type of analysis – most commonly applied to trypsin digests of proteins
in proteomic analyses [29] – takes the most abundant ion in a given mass spectrum and
fragments this ion, next the most abundant product ion is further fragmented generating
further structural data.
As the flavonoids are eluted from the HPLC column, they each -for a given time window-
become the most abundant such ion and so information on the majority of the flavonoids can
be obtained in an automated fashion.
Under MS/MS conditions, flavonoids are fragmented into a series of product ions. For a
flavonoid glycoside, the loss of the sugar moiety is commonly seen, producing the
corresponding aglycone as the main fragment ion. This is a very important characteristic and
can be used to facilitate the bioanalysis of individual flavonoid species in a mixture. For
flavonoid aglycones, the diagnostic product ions produced by further fragmentation are
illustrated in Figure 5.
Many of the modern mass spectrometers e.g. triple quadrupoles, Q-TOFs can carry out
MS/MS analysis since there have two tandem analysers, however ion trap mass spectrometers
have a unique capability in that it stores ions. Once an ion is stored, it can be manipulated in
many different ways to perform multistage MS/MS experiments (MSn), hence it is frequently
used for the structural elucidation of unknown flavonoids.
The application of this fragmentation process to the identification of a flavonoid
separated by the miniaturised HPLC separation and analysed by ESI-MS, MS/MS and MS3 is
shown in Figure 6 and Figure 7.
0,4B-
1,3A-
1,2A- R1
0,2 - R2
A
HO B
0 O 1
A
C 2
1,2B-
4 3 OH
OH O
0,4 -
A
Figure 5. Nomenclature and diagnostic product ions of deprotonated flavonols formed by ESI-ion trap
mass spectrometer, the symbols i,j A+ and i,jB+ are used to designate primary product ions containing
intact A and B rings, respectively. The superscripts i and j refer to the bonds of the C-ring that have
been broken [30].
310 Shujing Ding and Ed Dudley
Figure 6. Data-dependent analysis rutin by HPLC-MS showing the full scan mass spectrum, the
MS/MS spectrum of m/z 609 and the MS3 spectrum of m/z 301 via m/z 609.
The information obtained from the data dependent fragmentation analysis can also be re-
searched in order to study selectively specific types of flavonoid glycosides. This process is
based on the knowledge that under MS/MS conditions, the loss of the sugar moiety is the first
fragmentation event observed. The two most commonly bound sugars in these glycosides are
a glucosyl group and a rhamnosyl group.
Bioanalysis of the Flavonoid Composition of Herbal Extracts… 311
OH OH
OH
O- O-
-
O O
HO O HO O
-CO
OH
O OH O
OH
OH O OH O
m/z=301 m/z=273
CH3 -308 -COO
O
O HO
O OH
HO OH -
O -
O O
O
OH O -
O
C
OH O
OH OH O
OH
OH O
1,3 - 1,2 -
A A
Figure 7. Illustration of the MS/MS and MS3 fragmentation pathways observed during the
fragmentation of rutin in an ion trap mass spectrometer (see Figure 6).
When these are lost in MS/MS experiments the product ion formed represents the loss of
162Da and 146Da respectively. Therefore a useful approach is to highlight in the complex
dataset (obtained during the data dependent acquisition) all ions that exhibited these mass
losses during fragmentation analysis. Figure 8 indicates the total ion chromatogram for a
herbal extract indicating all ions monitored in the mass spectrometer compared to the
selecting highlighting of those that lose 162 and 146Da.
Figure 8. Capillary HPLC/MS total ion chromatogram (TIC) of Gingko biloba extract (a) base peak full
scan (b) neutral loss of m/z 162 (c) neutral loss of m/z 146.
312 Shujing Ding and Ed Dudley
It can clearly be seen that this filtering process allows the specific study of such
compounds and further study of these peaks confirmed their status as flavonoids.
Flavonoids show biological properties through their free radical-scavenging antioxidant
activities and metal-ion-chelating abilities [31]. Despite the benefits of these components,
their bioavailability after oral administration is considered to be a limiting factor [32]. Mass
spectrometry has therefore also been more recently applied to the analysis of flavonoids in the
urine of subjects who have taken herbal extracts in order to determine the time taken for the
compounds to be eliminated from the body. After ingestion, flavonoid glycosides are thought
to be first hydrolyzed by micro-organisms in the gastrointestinal tract to aglycones. The
liberated aglycones can be absorbed through the intestinal wall and are eventually excreted in
the urine and bile as glucuronides and sulphate conjugates [33]. The analysis of components
excreted in urine is complicated due to their lower levels and the large number of other high
abundant species also excreted. Therefore, the urine samples are generally incubated with the
enzyme β-glucuronidase/sulfatase to convert flavonoids into their aglycones as a first step of
the analysis. In doing so, aglycone concentrations are increased to a detectable level and the
analysis is simplified. A number of analytical techniques have been utilized in order to
evaluate the metabolism and bioavailability of flavonoids in vitro and in vivo. The methods
utilised previously for the study of urinary and other biologically important flavonoids levels
include HPLC [34-36] and mass spectrometry (GC/MS [37, 38] and HPLC/MS [39]). There
are also reports of online solid phase extraction using a reverse phase trap column (the outline
of such an experiment is shown in Figure 9 [40]) and this methodology was shown to allow
the quick and efficient clean up of the injected urine sample.
The comparison of the on-line clean-up method with off-line purification methodologies
demonstrated that the on-line purification protocol does not suffer from any significant loss of
analyte or interference from other matrix materials, and this method required minimal prior
sample preparation and thereby facilitating higher throughput and greater automation.
CONCLUSION
Flavonoids are a diverse, yet important, group of chemical entities in many herbal
extracts and their analysis in such materials is essential in order to gauge the appropriateness
of different commercially available extracts. However, such an undertaking is complicated by
the varied nature of these compounds. Fingerprint analysis – the comparison of the global
metabolite profile of an extract compared to a "pure" standard - offers the ability to monitor
such products and has been suggested as being beneficial by major regulatory organisations.
Whilst many different techniques have been applied to such analyses in the past, the
application of mass spectrometry offers an improvement in the quality of the data that can be
obtained in an automated fashion. Furthermore, the ease by which the flavonoid-specific data
can be extracted from the information (representing the total metabolite profile determined by
such analyses) is improved by such experiments.
Mass spectrometry has a further application in the quantitation of flavonoid elimination
via urinary excretion after the consumption of such herbal extracts and can therefore be of use
in determining the longevity of the flavonoid. The application of the on-line clean-up and
concentration of the flavonoids has been investigated and shown to allow accurate
quantitation whilst minimising sample pre-treatment and work-up prior to analysis.
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In: Flavonoids: Biosynthesis, Biological Effects… ISBN: 978-1-60741-622-7
Editor: Raymond B. Keller © 2009 Nova Science Publishers, Inc.
Chapter 13
Kola’ K. Ajibesin
Department of Pharmacognosy,
Olabisi Onabanjo University, Ogun State
ABSTRACT
Afrofittonia silvestris Lindau, commonly known as the hunter’s weed, is a
procumbent herb trailing on moist ground. The leaves of the plant are used to heal sore
feet, skin infections and as laxative. The leaves were macerated in 50 % ethanol and the
liquid extract concentrated to dryness. The dry extract was evaluated for antibacterial
activity by adopting agar diffusion method. The extract was partitioned between water,
ethyl acetate and butanol successively and further subjected to antibacterial testing. The
most active extract, ethyl acetate extract, was purified through various chromatographic
methods to obtain pure compounds identified by spectroscopic methods as kaempferide
3–O–β–D–glucopyranoside and kaempferol 5,4’-dimethoxy-3,7-O- α-L-dirhamnoside.
These compounds produced significant antibacterial effects, while the minimum
inhibitory concentrations of the fractions and the pure compounds ranged between 25 and
250 µg/mL. These flavonoids are reported for the first time in this plant, while
kaempferol 5,4’-dimethoxy-3,7-O- α-L-dirhamnoside is a new compound.
INTRODUCTION
Afrofittonia silvestris is a procumbent herb trailing on moist ground and commonly
known as the hunter’s weed (Hutchinson and Dalziel, 1958). The plant grows throughout the
year (Taylor, 1966). The leaves of the plant are employed in traditional medicine in the
treatment of diseases such as digestive tract disorders, sores, skin diseases and constipation
316 Kola’ K. Ajibesin
(Burkill, 1985; Etukudo, 2003; Ajibesin et al., 2008). Although no chemical report of this
plant is in literature, its nutritional values have been established (Odoemena et al., 2002).
This study was designed to isolate and identify the antibacterial principles of Afrofittonia
silvestris.
The NMR spectra were recorded on a Brucker DR – 500 MHz (1H 1) and 50 MHz (13C),
in CD3OD using TMS as internal standard. Mass spectroscopy was determined using Electro
spray ionization (ESI) Full MS and Finnigan LCQ Deca-MS, Agilent series 1100-LC. UV
spectroscopy was determined by Dionex, UVD 340 S Dionex. Melting points were
determined on a Kofler hot-stage microscope (uncorrected). TLC was carried out on silica gel
60 F254 (Merck). Solvent systems such as EtOAc-CH3OH; 8:2 (A), CH2Cl2-MeOH; 9:1 (B),
CH2Cl2-MeOH; 4:1 (C), CH2Cl2-MeOH; 7:3 (D), CH2Cl2-MeOH-H2O; (7:3:1) were
employed. UV light (λ max 254 nm and 366 nm), FeCl3 spray, vanillin/H2SO4 and conc.
H2SO4 sprays followed by activating at 100oC for 5 min., were used for detection of spots.
Plant Material
The leaves (6 kg) of Afrofittonia silvestris were collected in July, 2000, at Ikot Ekpene in
Akwa Ibom State, Nigeria. The plant was identified and authenticated by Dr. U. Essiett of the
Department of Botany, University of Uyo, Uyo, Nigeria. Voucher specimen (KKA 2) was
deposited in the Department of Pharmacognosy and Natural Medicine herbarium, Faculty of
Pharmacy, University of Uyo, Uyo, Nigeria.
The dried leaf powder (4 kg) of A. silvestris was extracted by maceration using 50 %
EtOH (10 L). It was filtered and the marc re-extracted with the fresh solvent mixture for 12 h
(x2) and filtered. The filtrates were pooled together and concentrated to dryness in vacuo at
40oC.
Antibacterial Test
The extracts and the fractions were reconstituted in MeOH-H2O (1:1) to obtain a stock
solution of 20 mg/mL. 50 µl of this solution was introduced into each of the equidistant wells
(8 mm) bored on the agar plate surface previously inoculated with each of the test organisms.
A control well containing Gentamicin (5 µg/mL) was placed in each of the plates seeded with
bacteria. The bacteria were incubated at 37 OC for 24 h (Alade and Irobi, 1993). Antibacterial
Antibacterial Effects of the Flavonoids of the Leaves of Afrofittonia Silvestris 317
The dry extract was dissolved in water and successively shaken with EtOAc (6x300 mL)
and BuOH (6x300 mL) to afford ethylacetate (20 g), butanol (25 g) and aqueous extracts (35
g) respectively.
The resultant 3 fractions were evaluated against the test organisms at 20 mg/mL in
aqueous methanol (MeOH:H2O; table 2). The antibacterial principles were partitioned more
largely into ethyl acetate fraction followed by butanol and aqueous fractions respectively.
The EtOAc, butanol and aqueous fractions of the plant species were subjected to TLC
analysis, using solvent systems A, D and E respectively, visualized under the UV light (λ 254
nm) before using 100 % H2SO4 and FeCl3 solution as detecting spray reagents. The most
active EtOAc fraction (16 g) showed flavonoid components and was chromatographed on
silica (Merck, 0.040-0.063 mm particle size) by accelerated gradient chromatography (AGC)
column and eluted with C6H14 containing increasing amount of CH2Cl2, followed by
increasing amount of CH3OH (4:1, 9:1). Four flavonoid fractions coded A, B, C, D were
obtained, two (B, C) of which showed significant antibacterial effects. The more active (C) of
the two flavonoid fractions was further fractionated on silica by vacuum liquid
chromatography (VLC), using EtOAc in gradient with CH3OH and H2O (7:2:1). Final
purification was carried out on silica by preparative thin layer chromatography (prep. TLC),
using EtOAc-CH3OH (4:1) as mobile phase to give 1 (30 mg). The less active flavonoid
fraction was purified by repeated AGC (silica) to yield 2 (21 mg). The two compounds along
with the fractions were subjected to antibacterial test.
kaempferol 5,4’-dimethoxy-3,7-O- α-L-dirhamnoside 1. Yellow powder, mp 220 0C
(MeOH), UV CH3OH λ max nm: 272, 334, ESI Full MS – m/z (rel. int.): 607 [M+H]+ (100),
461 [M+H-Rham.]+ (25), 299 [M+H-2Rham.]+ (5). 1HNMR (CD3OD): δ 6.70 (1h, d, J= 2.2
Hz, H-6), 6.77 (1H, d, J= 2.2 Hz, H-8), 7.10 (1H, d, J= 8.8 Hz, H-3’, 5’), 7.98 (1H, dd, J= 8.8
318 Kola’ K. Ajibesin
Hz, H-2’, 6’), 5.48 (1h, d, J= 1.4 Hz, H-1’’’), 5.24 (1H, d, J= 1.4 Hz, H-1’’’), 0.72 (3H, d, J=
6.2 Hz, H-6’’’), 0.64 (3H, d, J= 6.2 Hz, H-6’’), 3.29 (3H, s, -OMe), 3.95 (3H, s, -OMe), 2.40-
4.92 (8H, sugar protons).
13
CNMR: see table 1.
Known compound 2 was identified by comparing its spectroscopic data with those in
literature (Lahtinen et al., 2005).
et al., 1970). In addition, two anomeric protons assigned to H-1 of the two rhamnose moieties
were observed at δ5.24 and δ5.48 as narrow doublets for α configuration of the glycosidic
linkage (Toker et al., 2004).
The structure indicated by the 1HNMR was supported by 13CNMR spectrum. The two
methoxy groups attached at positions 5 and 4′ were indicated by the signals at 57.8 and 58.0
ppm respectively. The 13C NMR spectra gave many similarities with kaempferol 3,7-O- α-
dirhamnoside (Gohar and Elmazar, 1997) in the benzopyrone ring and sugar moiety.
However, the position of the 1st methoxy group of 1 was indicated by the downfield shift of
C-4´ by 2.7 ppm and the upfield shift of the ortho carbons (C-3´/5´) by 3.5 ppm with respect
to the same signals in the 13C NMR spectra of kaempferol 3, 7-O- α-dirhamnoside.
Furthermore, the position of the other methoxy group on C-5 was shown by the downfield
shift of C-5 by 2.3 ppm when compared with the 13C NMR signal of the same kaempferol
3,7-O- α- dirhamnoside, while the ortho carbon C-6 shifted much upfield by 5.2 ppm. Thus,
the main distinction between kaempferol 3,7-O- α- dirhamnoside and 1 appears to be the
methoxy groups present in the latter, which was further confirmed by its larger molar mass.
Similar distinctions were made when 1 was compared with another kaempferol 3,7- O-α-L-
dirhamnoside isolated from Tilia argentea (Toker et al., 2004). Also, 1 shared some
resonance characteristics when compared with the aglycone of kaempferide 3 rhamnoside
where only one methoxy was attached at C-4´ to form kaempferide (Bilia et al. 1993). The
glycosylation at C-3 and C-7 was shown by their 13CNMR signals at 134.8 ppm (s) and 164.0
ppm (s) respectively. The MS fragmentation of 1 also suggested O-glycosilation at C-3 and
C-7. The sugar region gave eight sugar carbon signals at ca. 70.1-72.0 ppm, with the
anomeric carbons resonating at 102.3 ppm (s, C-1′′) and 100.0 ppm (s, C-1′′′) respectively.
The presence of two characteristic rhamnose methyl signals at 17.9 and 18.0 ppm further
confirmed the presence of two rhamnose residues (Agrawal, 1989; Neeru et al., 1990).
Consequently, the data for compound 1 are uniquely consistent with the structure of
kaempferol -5,4′-dimethoxy-3,7-O- α-L-dirhamnoside, a compound hitherto not reported in
literature. The data showed that 1 is different from kaempferol-3,7- O- α-L-dirhamnoside
earlier isolated from Chenopodium species and Tilia argentea in that it has two extra
methoxyl groups (Gohar and Elmazar, 1997; Toker et al., 2004).
Kaempferide 3-O-β-glucoside 2, earlier detected in the larval faeces of sawfly species
(Lahtinen et al., 2005), was also present in the plant.
The antibacterial effects of A. silvestris have been determined to be due mainly to
kaempferol -5,4′-dimethoxy-3,7-O- α-L-dirhamnoside, while Kaempferide 3-O-β-glucoside
was also isolated as antibacterial principle showing less inhibitory activity. It is noteworthy
that kaempferol -5,4′-dimethoxy-3,7-O- α-L-dirhamnoside elicited better antibacterial effect
than Gentamicin, the standard drug used. Antimicrobial activity of phenolics has been
similarly established in Acalypha species (Adesina et al., 2000).
320 Kola’ K. Ajibesin
CONCLUSION
No previous report of the antibacterial effects of kaempferol -5,4′-dimethoxy-3,7-O- α-L-
dirhamnoside and Kaempferide 3-O-β-glucoside was found in literature. The two conpounds
are responsible for the antibacterial effects of the plant, and this validates its uses in
traditional medicine for treating infections.
Microorganism A B C D 1 2 Gen
E.coli NCIB 86 >250 250 200 >250 50 100 200
B. cereus
NCIB 6349 >250 200 100 >250 25 100 100
S. aureus
NCIB 8588 >250 250 100 >250 25 50 50
Ps. aeruginosa
NCIB 950 >250 250 100 >250 25 50 50
Gen: gentamicin.
Antibacterial Effects of the Flavonoids of the Leaves of Afrofittonia Silvestris 321
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In: Flavonoids: Biosynthesis, Biological Effects… ISBN: 978-1-60741-622-7
Editor: Raymond B. Keller © 2009 Nova Science Publishers, Inc.
Chapter 14
WHY IS BIOAVAILABILITY
OF ANTHOCYANINS SO LOW?
Sabina Passamonti∗
Department of Life Sciences, University of Trieste
Via L. Giorgieri 1, 34127 Trieste
Following ingestion, anthocyanins are detected intact in blood [5, 6] in a time lapse
considerably shorter than that observed with other dietary flavonoids [7]. However, the
anthocyanins concentrations in plasma barely exceed 10-7 M, which translates into less than
0.1% absorption, including anthocyanin metabolites [8]. These features are indicative of
various biochemical issues underlying the quite limited bioavailability of anthocyanins in
mammalian organisms.
∗
Email: spassamonti@units.it
324 Sabina Passamonti
2.2.3. Absorption of Anthocyanins into Intestinal Cell Monolayers: A Unique Role for
Bilitranslocase?
The occurrence of bilitranslocase on the apical domain of intestinal cells offers an
interpretation of data obtained testing anthocyanins absorption by human intestinal cell
(Caco-2) monolayers [25]. The data show a striking correspondence between the extent of
absorption of various anthocyanins and the affinity of anthocyanins for bilitranslocase [16].
Delphinidin 3-glucoside, the poorest anthocyanidin monoglucoside inhibitor of bilitranslocase
(Ki=8.6 µM), was the least absorbed compound; the most absorbed ones, peonidin 3-
glucoside and malvidin 3-glucoside, are also the best bilitranslocase ligands (Ki=1.8 and 1.4
µM, respectively). The correspondence also recurs with the glycosylated derivatives of
cyanidin: cyanidin 3-glucoside is better absorbed than cyanidin 3-galactoside and the former
is a better bilitranslocase ligand (Ki=5.8 µM) than the latter (Ki=35 µM).
The possible role of glucose transporters in anthocyanin translocation from the lumen
into intestinal cells has been ruled out on the basis that glucose fails to decrease cyanidin 3-
glucoside absorption both in vivo [26] and in in vitro intestinal models [27]. No experimental
data is currently available to put forward speculations about the possible involvement of other
apical transporters in anthocyanin absorption.
326 Sabina Passamonti
CONCLUSIONS
In conclusion, the poor bioavailability of anthocyanis seems to stem from the fact that
they are transported into intestinal cells by a carrier-mediated mechanism, that displays high
affinity though low capacity of transport. Contrary to many other organic anions, these
Why Is Bioavailability of Anthocyanins So Low? 327
pigments are apparently not transported by other intestinal membrane carriers besides
bilitranslocase, though this issue needs to be further investigated. Surprisingly, anthocyanins
are retained into the intestinal cells, being unable to be transported across the basolateral
domain of the enterocyte plasma membrane. The high intracellular concentration is certainly
a factor limiting sustained uptake of anthocyanin from the lumen. In the colon, anthocyanins
undergo de-glycosylation and rapid C-ring fission, perhaps catalysed by bacterial chalcone
isomerase. Enzyme-based degradation of anthocyanins in biological fluids is an issue that
deserves investigation, in order to assess all aspects of the limited anthocyanin bioavailability
in mammalian organisms.
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[22] Passamonti S, Vrhovsek U, Vanzo A, Mattivi F. The stomach as a site for anthocyanins
absorption from food. FEBS Letters 2003; 544: 210-3.
[23] Talavera S, Felgines C, Texier O, Besson C, Lamaison JL, Remesy C. Anthocyanins
Are Efficiently Absorbed from the Stomach in Anesthetized Rats. J. Nutr. 2003; 133:
4178-82.
[24] Karam SM, Leblond CP. Identifying and counting epithelial cell types in the "corpus"
of the mouse stomach. Anat Rec 1992; 232: 231-46.
[25] Yi W, Akoh CC, Fischer J, Krewer G. Absorption of anthocyanins from blueberry
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5651-8.
[26] Felgines C, Texier O, Besson C, Vitaglione P, Lamaison JL, Fogliano V, et al.
Influence of glucose on cyanidin 3-glucoside absorption in rats. Mol. Nutr. Food Res.
2008; 52: 959-64.
[27] Walton MC, McGhie TK, Reynolds GW, Hendriks WH. The flavonol quercetin-3-
glucoside inhibits cyanidin-3-glucoside absorption in vitro. J. Agric. Food Chem. 2006;
54: 4913-20.
[28] Steinert RE, Ditscheid B, Netzel M, Jahreis G. Absorption of black currant
anthocyanins by monolayers of human intestinal epithelial Caco-2 cells mounted in
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Flavonoid-mediated inhibition of intestinal ABC transporters may affect the oral
Why Is Bioavailability of Anthocyanins So Low? 329
agent, xii, 50, 159, 161, 183, 190, 194, 197, 199,
A 204, 207, 209, 211, 228, 291, 303
aggregation, 49, 163, 214
abatement, 159
aging, xii, 200, 201, 273, 274, 283, 287
accounting, 55
aging process, 283
accuracy, 306
agonist, 149, 238
acetic acid, 16, 31
AIDS, 142
acetone, 160, 252, 279
air pollutants, 159, 174
acid, ix, xi, 4, 5, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18,
albumin, 2, 22, 64, 146, 151
20, 21, 23, 26, 27, 29, 30, 31, 38, 41, 42, 43, 44,
alcohol, 12, 40, 156, 198, 252
47, 49, 55, 68, 90, 93, 97, 103, 104, 105, 112,
aldehydes, 105, 280
115, 145, 146, 148, 155, 157, 158, 160, 161, 162,
algae, 194
169, 174, 179, 188, 189, 190, 194, 195, 199, 201,
algorithm, 62, 67, 90
208, 225, 228, 245, 249, 251, 252, 253, 254, 255,
alkaloids, 292, 297
256, 258, 261, 278, 279, 280, 281, 282, 283, 284,
alkylation, 56
287, 288, 289, 296, 298, 303, 304, 325, 326, 328
alpha-tocopherol, 154
acidity, 60
alternative, 17, 19, 31, 62, 164, 234, 250, 304, 305
activation, xii, 25, 29, 30, 50, 59, 63, 91, 92, 149,
alternative medicine, 250
159, 173, 176, 183, 184, 186, 187, 189, 190, 191,
alternatives, xiii, 291
203, 205, 207, 209, 210, 214, 223, 224, 226, 234,
alters, 174, 175, 245
236, 238, 239, 243, 244, 245, 246, 247, 263, 264,
Alzheimer’s disease, xii, 63, 64, 214, 273, 274, 287
266, 267, 269, 270, 271, 272
amines, 156, 172
activation energy, 59
amino acids, 199, 306
active oxygen, 227, 244
ammonia, 195
active transport, 9, 13, 326
amplitude, 118, 120, 125
acute renal failure, 179
anaerobe, 16, 326
acylation, 292
analgesic, 226, 227, 250
adenocarcinoma, 65, 66, 209, 265, 267, 272
anaphylaxis, 171, 177
adenosine, 190
anastomosis, 260
adhesion, 25, 48, 206, 216
androgen, 210, 241, 269
adhesives, 117, 118
androgens, 235
adipose, 246
angiogenesis, 187, 206, 207, 242, 261, 264
ADP, 87
angiosperm, 292
adults, 214, 251
angiotensin II, 25, 48
Africa, 321
aniline, 87
agar, xiv, 315, 316
animals, 2, 24, 144, 145, 157, 158, 160, 201, 208,
age, vii, 201, 241
215, 217, 218, 219, 220, 221, 222, 223, 250, 251
ageing, 283
ANOVA, 217
332 Index
anthocyanin, xii, xiv, 6, 8, 9, 14, 15, 18, 19, 20, 21, availability, xiii, 22, 198, 199, 258, 291
23, 33, 45, 73, 74, 75, 87, 195, 263, 267, 323,
324, 325, 326, 327
antibiotic, 167 B
anti-cancer, 29, 201, 234
bacteria, 2, 5, 17, 18, 19, 44, 45, 164, 165, 171, 196,
anticancer activity, 88, 134, 197
316, 317
anticancer drug, 188, 190, 191, 197, 198, 209
bacterial infection, 63
anticoagulant, 86
bacterium, 44
antidepressant, 50
barriers, 61, 323
antigen, 171
BBB, 23, 24
anti-inflammatory drugs, 222, 228
Bcl-2 proteins, 266
antioxidant, vii, viii, x, xi, xii, 25, 30, 32, 34, 35, 43,
beer, 18, 43, 247
45, 46, 53, 54, 55, 59, 60, 61, 63, 67, 69, 80, 81,
behavior, 84, 91, 169, 206, 238
83, 85, 86, 89, 92, 93, 95, 98, 126, 127, 128, 130,
Beijing, 201
131, 132, 133, 141, 143, 144, 145, 146, 148, 151,
Belgium, 140, 216
152, 156, 158, 160, 161, 162, 163, 170, 173, 174,
beneficial effect, xi, 184, 213, 214, 274, 281, 287
175, 176, 177, 184, 195, 197, 199, 201, 203, 205,
benign, 183, 187
206, 207, 214, 225, 226, 227, 228, 232, 233, 234,
benzene, 65, 89
235, 240, 242, 244, 245, 247, 263, 267, 274, 278,
benzo(a)pyrene, 166
286, 288, 312
beta-carotene, 158, 162, 179
antioxidative activity, 161, 224
beverages, 167, 267, 287
antipyretic, 226
bias, 308
antitumor, 134, 191, 192, 210, 250
bile, 30, 64, 188, 225, 236, 312
antivenom, xii, 250
bile acids, 236
aorta, 28, 149, 172
bilirubin, 327, 328
apoptosis, xi, xii, 26, 30, 49, 51, 52, 64, 67, 68, 86,
binding, 2, 6, 22, 47, 48, 65, 66, 146, 151, 163, 173,
88, 90, 93, 153, 154, 155, 156, 157, 162, 163,
177, 186, 235, 236, 237, 238, 239, 245, 266, 280,
174, 175, 179, 180, 185, 186, 187, 188, 189, 190,
328
191, 192, 197, 204, 205, 206, 208, 209, 210, 211,
binding globulin, 151, 173
231, 234, 235, 239, 243, 244, 245, 247, 263, 264,
bioassay, 31, 85, 191
265, 266, 267, 268, 269, 270, 271, 272
bioavailability, vii, viii, x, 1, 6, 7, 8, 10, 13, 14, 15,
apples, 12, 18, 57, 58, 153
19, 20, 23, 30, 31, 33, 34, 35, 36, 40, 42, 45, 49,
aqueous solutions, 75, 293
53, 54, 63, 64, 70, 165, 166, 167, 168, 173, 180,
arabinoside, 175
181, 187, 188, 190, 191, 192, 198, 202, 207, 208,
aromatic compounds, 15, 42
210, 226, 245, 278, 312, 323, 326, 327, 329
arrest, 147, 155, 175, 176, 186, 188, 189, 190, 192,
biochemistry, 62, 89, 90, 296, 297, 298, 299
193, 203, 204, 205, 208, 210, 235, 266, 270, 271,
biodiversity, 296, 297
272
bioflavonoids, vii, 142, 151, 153, 164, 167, 170, 172,
arteries, 50
176
arthritis, x, 63, 181
bioinformatics, xiii, 291, 292
aryl hydrocarbon receptor, 184
biokinetics, 47
ascorbic acid, 161, 201
biological activity, viii, xi, 22, 24, 25, 28, 29, 30, 32,
Asia, 200
49, 53, 55, 62, 63, 82, 188, 200, 231, 261, 287
assessment, 296
biological processes, 239
asymmetry, 58, 64, 88
biological rhythms, 215
atherosclerosis, 28, 54, 63, 94, 143, 147, 244
biological systems, viii, 53, 55
atoms, 56, 62, 303, 306
biomarkers, 10, 21, 39, 46, 227, 228
ATP, 24, 235
biosynthesis, iv, 25, 48, 88, 143, 146, 150, 175, 195,
attacks, vii
296, 297
Australasia, 294
black tea, 42, 47, 268, 278, 287, 289
Australia, xi, 231, 232, 242, 294, 298
bladder, 264, 269
Austria, 216
blocks, 24, 164, 186, 242
automation, 313
Index 333
blood, 10, 18, 22, 23, 24, 25, 28, 39, 43, 46, 48, 63, carbohydrates, 103, 105, 117, 121, 124, 136, 214,
64, 89, 142, 143, 144, 146, 148, 151, 167, 176, 293
190, 193, 197, 214, 222, 224, 226, 228, 250, 302, carbon, 2, 31, 55, 198, 319
323, 326 carbon atoms, 198
blood flow, 144 carbon dioxide, 2, 31
blood pressure, 214, 222, 224, 226, 228 carcinogen, 26, 154, 184
blood vessels, 23 carcinogenesis, 156, 176, 183, 185, 187, 268
blood-brain barrier, 23, 48, 167, 176 carcinoma, 6, 51, 137, 190, 192, 193, 197, 203, 204,
bloodstream, 7, 14 205, 206, 209, 211, 264, 266, 271, 272
body weight, 51, 151 cardiac muscle, 201
bonds, 91, 275, 278, 309 cardiovascular disease, xi, xii, 2, 32, 50, 54, 214,
bone marrow, 156, 157, 159, 174 231, 233, 234, 273, 274, 287
bradykinin, xi, 213, 215, 217, 220, 221, 223, 224, cardiovascular risk, 33, 183
229, 250 cardiovascular system, x, 141, 142, 214
brain, 8, 10, 11, 23, 24, 26, 28, 30, 37, 38, 48, 50, carotene, 158
161, 167, 214 carotenoids, 153, 179
branching, 110 carrier, 13, 22, 24, 48, 64, 136, 198, 199, 324, 325,
Brazil, 109, 249, 251, 252, 253, 261 326, 327, 328
breakdown, 15, 19, 142 caspases, 186, 247, 269
breast cancer, 6, 13, 24, 152, 154, 179, 183, 188, catalyst, 278
189, 195, 202, 204, 205, 206, 208, 235, 241, 243, catalytic activity, 256
246, 264, 265, 266, 269, 270, 271, 272, 302 cation, 58, 59, 74, 75, 94, 157, 170
breast carcinoma, 152, 187, 205, 207, 265, 269, 271 C-C, 58, 275, 281
breeding, 242 CD95, 186, 209, 211
bronchitis, 281 cDNA, 203, 207
buffer, 23, 25, 65, 66, 216, 252 cell culture, 13, 24, 25, 26, 31, 34, 51, 65, 135, 136,
building blocks, 58, 100 137, 138, 191, 219, 234, 267
cell cycle, 147, 176, 185, 186, 187, 188, 189, 190,
191, 193, 203, 204, 205, 208, 210, 264, 265, 266,
C 270, 272
cell death, 27, 50, 63, 64, 66, 67, 68, 69, 73, 85, 88,
Ca2+, 65
94, 95, 155, 172, 185, 186, 192, 234, 238, 244,
cabbage, 266
245, 271, 272
caecum, 15, 16, 17, 44
cell fate, 264, 270
caffeine, 142, 165, 173, 224
cell invasion, 207, 235, 243
calcium, 65, 142, 148, 165
cell killing, 87
calcium channel blocker, 142
cell line, viii, 51, 53, 54, 85, 147, 148, 152, 153, 167,
calorimetry, 60
175, 186, 187, 189, 190, 192, 202, 203, 204, 205,
Canada, 35
206, 209, 210, 222, 225, 242, 243, 265, 266, 267,
cancer, x, xi, xii, 2, 29, 32, 36, 38, 40, 47, 54, 63, 91,
269, 271
142, 152, 153, 154, 175, 181, 183, 185, 186, 187,
cell lines, viii, 51, 53, 54, 85, 153, 175, 186, 187,
188, 189, 190, 193, 194, 196, 197, 198, 200, 201,
189, 190, 192, 203, 204, 205, 209, 210, 243, 265,
203, 204, 206, 207, 231, 232, 233, 234, 235, 237,
266, 269, 271
239, 242, 244, 245, 247, 263, 264, 265, 266, 267,
cell membranes, 7, 30, 81, 88
268, 269, 270, 272, 273, 274, 287
cell metabolism, 14, 15
cancer cells, xii, 29, 185, 187, 188, 190, 197, 200,
cell signaling, xi, 63, 232, 239
204, 239, 244, 247, 263, 264, 265, 266, 267, 269,
cellular homeostasis, 65
270, 272
central nervous system, 23, 24, 28, 89, 193, 200,
cancer progression, 235
201, 226
candidates, 223
cerebellum, 24
capillary, 167, 250, 304
cerebral cortex, 24
capsule, 165
cervical cancer, 197
carbohydrate, ix, 98, 121, 124, 125, 199
channels, 149
334 Index
crystallization, 198, 261 diabetes, 2, 32, 151, 152, 177, 197, 199, 200, 201
cultivation, xiii, 301 diabetic nephropathy, 282, 289
culture, 48, 49, 89, 195, 196, 217, 218, 267, 317 diaphragm, xi, 249, 250, 252, 255, 257, 259, 260
culture media, 317 diastolic blood pressure, 145
curing, 134 diet, viii, x, xi, xiv, 8, 10, 11, 12, 17, 18, 19, 21, 22,
CVD, 26, 29, 31 23, 28, 31, 53, 57, 63, 142, 144, 145, 146, 161,
cycles, 60 162, 175, 179, 181, 182, 189, 213, 215, 221, 222,
cyclins, 147, 155 224, 236
cyclooxygenase, 48, 50, 225 dietary intake, xii, 5, 19, 31, 232, 263, 289
cytochrome, 14, 156, 165, 166, 172, 173, 179, 190, dietary supplementation, 11, 34
205, 234, 237, 244, 247 differentiation, 192, 239
cytochrome p450, 190 diffusion, xiv, 6, 7, 12, 13, 18, 22, 24, 30, 48, 61, 87,
cytokines, 190, 215, 222, 224, 227 315, 326
cytometry, 65, 66, 69, 94 diffusion rates, 87
cytosine, 175 digestion, 3, 6, 34, 198, 302, 329
cytoskeleton, 155 dilation, 29, 32
cytotoxic agents, 185 dimerization, 50, 237
cytotoxicity, 26, 27, 30, 89, 156, 157, 163, 175, 177, dislocation, 75
190, 197, 208 displacement, 253
disposition, 34, 45, 169, 170
dissociation, viii, 53, 59, 60, 80, 81, 84, 87, 91, 94,
D 237, 239, 266
distilled water, 217
data analysis, 62
distribution, ix, xii, 2, 3, 14, 22, 34, 35, 37, 38, 46,
death, 23, 28, 67, 68, 186, 190, 205, 209, 210
56, 57, 64, 82, 94, 97, 105, 117, 187, 192, 245,
decay, vii, ix, 98, 126, 127, 128, 129, 130, 131, 132,
273, 292, 293, 299, 325
133
diversity, xi, 54, 60, 94, 159, 249, 293
decomposition, 103
division, 192, 293
decoupling, 239
DNA, 55, 62, 86, 87, 89, 93, 153, 155, 156, 157,
defects, 148
158, 160, 161, 163, 177, 178, 179, 183, 184, 185,
defense, 156, 162, 214
186, 192, 194, 195, 234, 236, 237, 238, 244, 247,
deficiency, 58
292, 296, 297
degradation, 2, 9, 11, 14, 15, 16, 19, 21, 22, 30, 45,
DNA damage, 86, 89, 156, 157, 158, 160, 161, 186,
55, 62, 89, 93, 103, 127, 187, 198, 235, 264, 278,
192
280, 281, 286, 326, 327, 329
DNA sequencing, 292
delivery, 178, 198, 199
DNA strand breaks, 179
density, 51, 61, 65, 66, 67, 86, 87, 89, 95, 148, 227,
dopaminergic, 225
325
dosage, 68, 168
density functional theory, 61, 86, 95
dose-response relationship, 67
dental caries, 164, 179
dosing, 6, 7, 9, 18, 21, 45
dependent variable, 217
down-regulation, 206, 209, 224, 225, 264, 265
dephosphorylation, 271
drug action, 245
depolarization, 190
drug discovery, 91, 327
depolymerization, 103, 105, 289
drug efflux, 13, 22
deregulation, 265
drug interaction, x, 141, 142, 166, 169, 173, 176,
derivatives, viii, 2, 7, 8, 13, 14, 16, 20, 21, 27, 28,
207, 245
44, 60, 63, 80, 89, 97, 150, 151, 152, 171, 191,
drug metabolism, 190, 203, 237
192, 193, 194, 197, 201, 214, 246, 270, 278, 279,
drug resistance, 6, 22, 264
280, 288, 296, 325
drug use, 190, 319
detachment, ix, 97, 117
drugs, x, xiii, 24, 141, 142, 155, 167, 169, 181, 183,
detection, 14, 86, 93, 293, 294, 297, 298, 305, 316
188, 193, 197, 199, 200, 201, 202, 211, 237, 250,
detection techniques, 294
261, 301, 303, 329
developed countries, 287
drying, 178
DFT, 61, 93, 94, 95
336 Index
duodenum, 64, 208 estrogen, xi, 142, 189, 195, 204, 231, 235, 237, 238,
duration, 15, 185 241, 242, 243, 244, 245, 246, 247, 248, 269, 271
dyes, 118 ethanol, xiv, 148, 150, 156, 176, 178, 184, 279, 315
ethanol metabolism, 156, 178
ethers, 25
E ethyl acetate, xiv, 148, 279, 315, 317, 320
ethylene, 217
E.coli, 216, 320
EU, 246
East Asia, 109
eucalyptus, 195
ECM, 187
Europe, 304
ecology, 297
euthanasia, 23, 24
edema, xi, 213, 220, 221, 222, 223, 224, 226, 227,
evaporation, 279
228
evolution, 183, 187, 221, 287
efflux transporters, 22, 23, 325, 326
excitation, 216
Egypt, 321
exclusion, 65, 293
elasticity, 118
excretion, 2, 6, 8, 10, 11, 14, 15, 19, 20, 21, 30, 31,
elderly, 38, 46
34, 35, 37, 39, 40, 42, 45, 46, 166, 187, 224, 282,
electrodes, 253
313
electron, 58, 59, 73, 75, 129, 130, 146, 177
execution, 163
electronic structure, 92
experimental condition, 30
electrophoresis, 304, 305
experimental design, 215, 253, 258
ELISA, 149, 154, 216, 219
expertise, 296
embryonic stem cells, 207
exposure, 33, 38, 42, 64, 68, 85, 90, 93, 158, 159,
emission, 216
168, 173, 183, 188, 190, 233, 241, 243
emulsions, 92
extracellular matrix, 187
encoding, 195
extraction, 14, 20, 196, 250, 252, 279, 304, 305, 312
endocrine, x, xi, 22, 141, 150, 231, 239, 241
endocrine system, x, 141, 150, 241
endocrine-disrupting chemicals, xi, 231 F
endothelial cells, 23, 25, 48, 143, 167, 206, 223, 228,
236, 238, 246, 248 fabrication, 116
endothelial dysfunction, 32 failure, 31, 187
endothelium, 149, 226 family, xii, 55, 58, 185, 186, 263, 264, 266, 268,
energy, 59, 70, 80, 84 269, 292, 294
England, 65, 260, 297, 298 fat, 51, 146, 175, 182, 197
enthusiasm, 292 fatigue, 214
environment, x, 14, 54, 61, 68, 141, 155, 299 fatty acids, 145, 196, 197, 200, 281
enzymatic activity, 142 FDA, 303
enzymes, vii, xi, 6, 63, 64, 84, 144, 146, 151, 153, feedback, 241
157, 158, 162, 174, 175, 177, 183, 184, 186, 195, feet, xiv, 315
198, 203, 232, 234, 235, 237, 260, 324 femur, 148
epidemiologic studies, 182, 263 fermentation, 44, 51, 278, 281, 283
epithelia, 15, 325, 328 fever, 214
epithelial cells, 5, 7, 15, 25, 27, 41, 64, 150, 173, fibroblasts, 25, 28, 48, 50
179, 205, 206, 243, 245, 265, 271, 323, 325 filtration, 306
epithelium, 324, 325, 326 financial support, 296
equating, 23 Finland, 183
equilibrium, 61, 74, 75, 87, 117 fission, 326, 327
erythrocyte membranes, 160, 172 flavor, x, 141
erythrocytes, 156 flight, viii, 97, 99
ESI, 163, 281, 305, 306, 309, 316, 317, 318 flora, 4, 6, 8, 9, 13, 16, 17, 18, 19, 21, 30, 44, 63, 326
ESR, 126, 127, 128, 130, 131, 133 fluid, 11
ester, 145, 197, 199, 252 fluorescence, 45, 47, 92, 216
estimating, 91 focusing, 254
Index 337
inflammation, x, xi, 29, 63, 87, 91, 92, 170, 181, isolation, xii, 216, 250, 254, 258, 273, 274, 286, 293,
193, 194, 196, 197, 200, 213, 215, 217, 220, 221, 294, 295, 309
222, 223, 225, 227, 228, 231, 245 isomerization, 191
inflammatory mediators, x, 213, 215, 222, 223, 224, isomers, 4, 14, 22, 26, 36, 107, 192, 275, 280, 285
227, 228 Israel, 195
inflammatory responses, 90 Italy, 32, 139, 215, 217, 231
ingest, 267 IV collagenase, 206
ingestion, 8, 12, 37, 38, 40, 46, 47, 48, 156, 233,
312, 323
inhibition, 20, 25, 26, 27, 29, 30, 48, 55, 67, 86, 95, J
127, 142, 143, 144, 145, 147, 148, 149, 150, 153,
Japan, 263, 273, 279, 281
154, 155, 157, 159, 160, 162, 163, 166, 167, 168,
jejunum, 9, 15, 37
169, 173, 174, 176, 177, 178, 179, 184, 185, 186,
187, 188, 191, 192, 193, 195, 196, 197, 200, 201,
205, 206, 208, 210, 211, 219, 220, 222, 223, 224, K
225, 234, 235, 239, 240, 244, 246, 248, 264, 265,
268, 269, 271, 272, 274, 317, 320, 324, 328 K+, 55, 91, 149, 178
inhibitor, 24, 25, 26, 148, 150, 151, 165, 166, 167, keratin, 155
168, 172, 177, 197, 204, 238, 261, 265, 288, 325 kidney, 8, 10, 11, 37, 161, 178, 179, 282, 289, 302
initiation, 183, 184, 202, 233, 235 kinase activity, 235, 265
injections, 223 kinetic studies, 47
injuries, 227 kinetics, ix, 46, 60, 87, 98, 128, 129, 130, 133, 324
innovation, 193, 194, 196, 198
inositol, 235
instability, 19, 75, 199, 326 L
instruction, 66
instruments, 60 lactase, 7, 12, 35, 40
insulin, 151, 201, 226, 234, 235, 246, 266 lactate dehydrogenase, 146
insulin resistance, 201, 226 language, 197
integration, 244 LDL, 22, 25, 47, 55, 93, 143, 144, 145, 162, 175,
integrity, 64 177, 202, 224, 238, 243, 244, 274
interaction, xi, 35, 61, 62, 64, 92, 121, 124, 142, 151, learning, 233
158, 163, 165, 166, 169, 172, 173, 179, 184, 199, leptin, 151
232, 237, 238, 241, 324, 328 lesions, 28, 176
interactions, ix, xi, 34, 54, 84, 89, 98, 121, 124, 125, leucine, 184
163, 165, 186, 206, 231, 233, 243, 287 leukemia, 49, 137, 156, 157, 205, 209, 210, 211,
intercellular adhesion molecule, 143, 173 247, 267, 272
interface, 23, 67, 82, 83 Lewis acids, 99, 126
interference, 192, 239, 258, 313 liberation, 18
interferon, 227 lifestyle, xii, 273, 274, 286
intervention, viii, 2, 29, 33, 187 ligand, 48, 163, 184, 186, 190, 209, 210, 236, 237,
intestinal tract, 18, 44, 325 243, 245, 266, 325
intestine, 5, 6, 7, 8, 9, 10, 11, 12, 14, 18, 30, 36, 40, lignans, 10, 161
44, 63, 64, 152, 158, 326 linkage, ix, 57, 98, 132, 200, 275, 278, 319
ionic polymers, 121 links, 105, 132
ionization, 59, 99, 228, 316 lipid metabolism, 51, 156, 175, 237
ions, 306, 308, 309, 311 lipid peroxidation, 11, 48, 55, 91, 153, 158, 160, 161,
Ireland, 297 172, 178, 245
iron, 146, 158, 174 lipid peroxides, 146, 147, 177, 178
irradiation, 63, 99, 126, 127, 128, 133, 160 lipids, 51, 145, 146, 147, 174, 177, 178, 214, 234
ischemia, 64, 90, 144, 179, 201 lipoproteins, 22, 40, 177, 326
isoflavonoid, 35, 195, 270 liquid chromatography, 14, 39, 40, 46, 86, 293, 304,
isoflavonoids, 43, 238, 242 305, 317, 318
liquids, ix, 89, 98, 118, 125
340 Index
liver, 7, 8, 10, 11, 14, 22, 26, 30, 35, 36, 38, 63, 64, mediation, 271
87, 143, 155, 156, 158, 165, 166, 167, 172, 175, medication, 142
179, 197, 208, 209, 236, 238, 244, 302, 324, 327, MEK, 51
328 melanoma, 204, 207, 208, 209, 242
liver cancer, 197 membranes, 48, 59, 61, 64, 82, 84, 95, 134
liver damage, 26 memory, 38
liver enzymes, 143 men, 9, 165, 202
localization, 238, 241, 328 messengers, 92, 233
location, viii, 53, 54, 84, 85 meta-analysis, 33
locus, 61 metabolic pathways, 55
longevity, 87, 313 metabolic syndrome, xii, 273, 274, 286, 287
lovastatin, 143, 144, 145, 154, 161, 169, 174 metabolism, iv, vii, viii, 1, 2, 3, 5, 6, 7, 8, 10, 13, 14,
low-density lipoprotein, 49, 92, 144, 162, 233 16, 18, 19, 21, 22, 27, 28, 29, 31, 32, 33, 34, 35,
LTD, 298 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
luciferase, 238 50, 53, 54, 55, 62, 63, 84, 85, 87, 88, 93, 142,
lumen, 6, 7, 9, 11, 12, 13, 14, 36, 64, 323, 325, 327 143, 156, 166, 168, 172, 173, 178, 179, 180, 183,
lung cancer, 32, 153, 176, 183, 202, 204, 209, 265, 187, 190, 203, 209, 225, 235, 237, 243, 312
267, 270, 271 metabolites, vii, viii, 1, 2, 3, 4, 8, 9, 10, 12, 14, 16,
luteinizing hormone, 233 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31,
lymph, 40, 329 36, 37, 38, 39, 40, 42, 43, 45, 46, 47, 48, 49, 50,
lymphatic system, 326 51, 52, 55, 56, 63, 93, 166, 168, 171, 175, 187,
lymphocytes, 28, 50, 51, 203, 205, 225 190, 195, 222, 225, 246, 247, 283, 292, 323, 326,
lymphoma, 190, 209 329
lysine, 199, 201 metabolizing, 183, 186
metastasis, 187, 201, 264
methanol, 133, 200, 201, 226, 252, 254, 279, 280,
M 286, 295, 306, 317
methyl groups, 14, 31, 56
macrophages, x, 27, 28, 49, 159, 175, 213, 215, 216,
methylation, x, 2, 9, 13, 14, 25, 29, 50, 58, 63, 182,
217, 218, 219, 222, 223, 224, 227
191, 202
magnesium, 75
Miami, 66
Malaysia, 139
mice, 30, 37, 51, 144, 145, 153, 155, 156, 157, 158,
males, 233
159, 160, 167, 172, 174, 175, 188, 206, 207, 208,
malignancy, 92
223, 244, 250, 251, 252, 254, 261
management, 148
microdialysis, 90
manipulation, 71
micrograms, 152
manufacturing, 214, 227
microgravity, 49
market, xi, 231, 233
micronucleus, 156, 159, 174
Mars, 198
micronutrients, 183
mass loss, 311
microscope, 316
mass spectrometry, xiii, 39, 40, 46, 60, 86, 99, 163,
microsomes, 156, 165, 167, 209
293, 296, 302, 304, 305, 308, 312, 313
microstructure, 121
mast cells, 223, 227
microstructures, ix, 98, 121, 125
matrix, 11, 34, 99, 148, 173, 187, 206, 207, 235, 243,
migration, 132, 150, 173, 216, 222, 243
313
milk, 19, 228
matrix metalloproteinase, 173, 187, 206, 207, 243
milligrams, 280
maturation, 186, 283
mineral water, 215
MCP, 143, 176, 197, 215
minority, 100
MCP-1, 143, 176, 197, 215
mitochondria, 54, 161, 192
meals, 237, 241
mitogen, 184, 238, 264
measurement, viii, 53, 60, 64, 84, 127
mitosis, 192
measures, 23, 118
mixing, 200
mechanistic explanations, 84
MMP, 187, 207, 243
media, 58, 65, 74, 87, 216, 293, 295
Index 341
124, 125, 191, 226, 273, 275, 278, 279, 280, 281, periodontal, 165, 179
284, 285, 286, 288, 289 peripheral blood, 30, 190, 193, 215, 226, 302
olive oil, 42 peripheral blood mononuclear cell, 30, 190, 193,
oncogenes, 264 215, 226
optimization, 67, 70, 199 peritoneal cavity, 216
oral cavity, 5, 34 peritonitis, 170, 176
orange juice, 10, 18, 37, 39, 43, 152 permeability, 6, 12, 23, 48, 62, 91, 169, 191, 222,
organ, 14, 23, 30, 237, 252 327
organelles, 84 permeation, 167
organic chemicals, 61 permit, 317
organic compounds, vii, 60, 92 peroxidation, 25, 95, 160, 161
organism, x, 181, 190 peroxide, 64, 65, 68, 86, 87, 247
orientation, 63, 307 peroxynitrite, 55, 88
osteoporosis, xi, 148, 179, 231, 233, 302 personal communication, 239
ovarian cancer, 154, 176, 184, 203, 206 pH, viii, ix, 14, 41, 53, 58, 75, 90, 98, 121, 124, 125,
ovariectomy, 51, 149 216, 252, 253, 281, 324, 326
oxidation, 17, 22, 25, 40, 47, 49, 55, 63, 72, 73, 78, pharmacokinetics, x, 22, 33, 39, 40, 141, 165, 166,
88, 89, 92, 93, 143, 145, 157, 158, 171, 177, 183, 167, 168, 172, 174, 175, 176, 208
198, 202, 228, 234, 243, 274, 278, 280, 281 pharmacology, 93
oxidation rate, 78 phenol, 87, 89, 94, 171, 191, 201, 216
oxidative damage, 39, 49, 82, 85, 174 phenoxyl radicals, 73, 126, 127, 171, 234
oxidative stress, 26, 27, 28, 50, 54, 63, 64, 85, 88, phenylalanine, 55, 56, 195
89, 144, 157, 158, 225, 227, 233, 245 phosphatidylserine, 64, 93
oxidizability, 78 phosphoenolpyruvate, 151
oxygen, xi, 22, 56, 57, 58, 62, 64, 89, 92, 93, 115, phospholipids, 64, 145
128, 129, 131, 132, 144, 163, 171, 197, 213, 222 phosphorylation, 49, 187, 188, 234, 235, 239, 246,
248, 264, 266, 267
phylum, 194
P physical properties, 55
physicochemical properties, viii, 53, 55, 65, 84, 85
p53, xii, 147, 155, 188, 189, 204, 208, 210, 211, 263,
physiology, 7, 62, 241
264, 265, 266, 267, 269, 270, 271, 272
pigs, 6, 7, 11, 20, 23, 34, 35, 38, 45, 170
PAA, 216
pilot study, 50
paclitaxel, xiii, 167, 168, 173, 176, 190, 209, 291
pith, 117, 126
pain, 214, 224, 302
placebo, 46, 225
Panama, 32
plants, vii, xii, xiii, 3, 4, 18, 22, 54, 55, 56, 57, 87,
pancreatic cancer, 205
90, 94, 177, 194, 195, 196, 232, 250, 251, 254,
paralysis, 254, 256
256, 258, 260, 261, 262, 263, 273, 280, 286, 287,
parameter, 60, 61, 62, 84, 91, 94, 127, 188, 304
288, 291, 292, 296, 298, 299, 301, 302, 303, 321
Parkinson’s disease, 64, 214
plasma, 2, 6, 7, 8, 9, 10, 11, 12, 14, 15, 18, 21, 22,
partition, viii, 53, 59, 61, 83, 84, 87, 88, 89, 91, 94
23, 24, 25, 28, 30, 33, 39, 40, 46, 48, 51, 64, 70,
passive, 5, 6, 7, 12, 13, 18, 22, 48, 61, 171
90, 143, 144, 145, 146, 148, 151, 157, 161, 162,
pasture, 242
167, 168, 174, 175, 176, 187, 190, 207, 214, 224,
patents, x, 181, 193, 196, 200, 202
236, 237, 240, 241, 244, 323, 324, 325, 326, 327,
pathogens, 165, 179
328
pathways, vii, x, 1, 15, 54, 85, 181, 183, 184, 185,
plasma levels, 18, 23
186, 192, 193, 205, 223, 225, 235, 237, 239, 241,
plasma membrane, 64, 190, 237, 324, 325, 327, 328
243, 245, 264, 265, 267, 268, 271, 272, 311
plasma proteins, 326
PBMC, 193
platelet aggregation, 26, 28, 50, 274
PCA, 9, 18, 20, 26, 27, 171
platelets, 49, 189
PCR, 149, 159
PLS, 92
penicillin, 66, 216
PM3, 61, 67, 70, 80
peptides, 87, 306
PM3 method, 61
perfusion, 9, 23
Index 343
polarity, 61 prostate cancer, 30, 51, 186, 200, 204, 205, 206, 207,
pollen, xiii, 301 210, 246, 265, 266, 267, 270, 271, 272
pollution, 47 prostate carcinoma, 203, 264, 265, 269, 270, 271
polycondensation, 105 protective role, 146, 152, 159
polymer, 200, 286 protein kinase C, 86, 235, 238, 264
polymerase, 190 protein kinases, 184, 244, 264
polymerization, 100, 103, 105, 107, 109, 118, 198, protein sequence, 326
280, 281, 285 proteins, xii, 2, 14, 22, 23, 30, 48, 62, 133, 136, 146,
polymers, x, xii, 5, 6, 14, 16, 58, 63, 118, 132, 213, 186, 190, 203, 209, 214, 234, 235, 237, 238, 239,
214, 273, 275, 278, 280, 281, 284, 285 250, 252, 258, 261, 264, 266, 268, 269, 271, 272,
polypeptide, 168, 172 274, 280, 287, 293, 306, 309, 328
polyphenols, x, 10, 11, 32, 33, 36, 37, 40, 41, 42, 45, protocol, 251, 252, 254, 305, 313
50, 51, 63, 86, 90, 92, 153, 181, 182, 183, 186, protons, 9, 318
187, 188, 189, 190, 193, 194, 196, 197, 198, 199, prototype, 250
200, 201, 202, 205, 207, 214, 226, 228, 234, 242, Pseudomonas aeruginosa, 164
263, 268, 281, 282, 289, 327 pulmonary edema, 170, 176
poor, 144, 187, 192, 237, 326 pumps, 2, 6, 13, 20, 22, 24, 26, 30
population, 32, 65, 153, 162, 183, 187, 215, 225, purification, xii, 117, 203, 250, 279, 313, 317
237, 263, 267 pylorus, 150
population growth, 162 pyrylium, 75
Portugal, 211
potassium, 306
potato, 8, 37, 267, 272 Q
power, 87, 127, 133, 221
quality control, 304
precipitation, xii, 249, 256, 261, 288
quantum chemical calculations, 61
predictability, 172
quartz, 127, 128, 129, 130, 131
prediction, 34, 86, 325
query, 261
preference, 24, 238
quinone, 17, 73, 86, 87, 184, 185, 203
pressure, 23, 279
prevention, xii, xiii, 26, 29, 47, 89, 94, 154, 188,
200, 201, 203, 206, 237, 242, 263, 265, 268, 270, R
273, 274, 287, 301, 302
primary cells, 54 race, 198
probe, 165, 216 radiation, 156, 157, 158, 174, 260
prodrugs, 169 radical formation, ix, 80, 98, 126, 127, 128, 129,
producers, 29, 194 131, 132, 133
production, vii, 27, 48, 52, 64, 155, 159, 175, 183, radical mechanism, 89
186, 189, 193, 194, 195, 196, 197, 199, 201, 208, radical reactions, 133
214, 215, 216, 218, 222, 223, 227, 233, 234, 235, radio, 12
238, 245 range, viii, ix, xiii, 7, 10, 24, 53, 54, 62, 64, 66, 69,
progesterone, 195 85, 98, 99, 102, 104, 108, 112, 121, 124, 125,
program, 61, 82, 83, 88, 279 158, 167, 215, 240, 281, 291, 305
pro-inflammatory, 183, 222 raw materials, 193
proliferation, x, xi, 55, 137, 147, 149, 152, 154, 156, reactive oxygen, 25, 48, 62, 81, 208, 222, 224, 233,
176, 179, 182, 185, 188, 190, 191, 192, 195, 196, 245
197, 200, 201, 202, 204, 205, 208, 210, 225, 231, reactivity, 64, 77, 82, 84, 235, 289
239, 244, 245, 246, 266, 271, 272 reagents, 278, 317
promoter, 194, 239 reality, 124
proposition, 31 receptors, xi, 22, 190, 209, 231, 233, 236, 237, 242,
prostaglandins, 150, 223 243, 244, 245, 247
prostate, 26, 30, 51, 186, 191, 200, 203, 204, 205, recognition, 328
206, 207, 210, 235, 242, 245, 246, 264, 265, 266, recovery, 144, 158, 173
267, 269, 270, 271, 272 recycling, 13, 34, 41, 64
344 Index
smooth muscle, x, 141, 147, 149, 172, 176, 178, 243, strain, 16, 118, 120, 121, 124, 125, 164
245, 272 strength, 22, 60, 84, 94, 148
smooth muscle cells, 147, 149, 178, 245 stress, 26, 28, 50, 64, 93, 196, 238
snake venom, 250, 251, 254, 256, 260, 261, 262 stressors, 54
snakes, 250, 251, 261 stroke, 2, 32
sodium, 13, 40, 42, 75, 101, 210, 252, 280, 306 structural characteristics, 327
software, 217 structural modifications, 117
soil, 57 structural transformations, 75, 87
solid phase, 305, 312 subjective judgments, 304
solubility, 11, 59, 65, 81, 82, 89, 90, 91, 107, 199, substitutes, 73
327 substitution, 73, 75, 81, 83, 161
solvation, ix, 94, 98, 133 substrates, 22, 167, 169, 186, 228
solvents, 61, 65, 133, 254 subtraction, 58
South Africa, 140 sucrose, 179
soy bean, xiii, 301 sugar, xiii, 30, 35, 45, 56, 57, 75, 77, 78, 83, 89, 296,
soybean, 38, 51, 265 301, 303, 309, 310, 318, 319
soybeans, 196 Sun, 80, 93, 94, 95, 205, 207, 209, 287, 288, 314
Spain, 213, 215, 224 suppression, 85, 154, 155, 158, 183, 264, 266, 269,
species, ix, xi, xiii, 3, 25, 48, 60, 62, 64, 68, 80, 81, 308
88, 89, 92, 97, 98, 109, 110, 121, 127, 132, 144, surface area, 91
163, 194, 201, 208, 213, 222, 224, 233, 245, 250, surfactant, 117
286, 291, 292, 293, 294, 295, 297, 298, 301, 309, surprise, 193
312, 317, 319, 321, 324 survival, xii, 26, 49, 56, 159, 174, 189, 211, 225,
specificity, 24, 245, 305 247, 263, 265, 266, 278
spectrophotometry, 59, 304 survival rate, 189
spectroscopy, viii, 14, 47, 97, 293, 294, 316 susceptibility, 40, 107, 143, 177, 227
spectrum, 88, 99, 102, 103, 104, 108, 110, 112, 116, swelling, 30
233, 239, 254, 306, 307, 308, 309, 310, 318, 319 switching, 312
speed, 304 Switzerland, 65, 216, 252
spermatogenesis, 233 symbols, 309
spin, 92, 126, 129, 130 symptoms, 29, 232
spleen, 10 synapse, 250
Sprague-Dawley rats, 51, 144, 146, 152, 170, 243, synergistic effect, x, 182, 183, 201, 202
244 synthesis, 55, 149, 193, 194, 196, 197, 199, 203,
SPSS, 217 210, 222, 223, 235, 246
squamous cell, 205, 269 systems, 8, 33, 55, 86, 92, 93, 170, 234, 256, 279,
squamous cell carcinoma, 205, 269 308, 316, 317
stability, 6, 22, 36, 41, 58, 60, 73, 80, 84, 88, 132,
198, 199, 283, 292
stabilization, 80, 87, 131, 199 T
stages, 3, 66, 183, 186
T cell, 27, 226
standard deviation, 68, 69
T lymphocytes, 30, 207
standards, 252, 254, 261
tannins, viii, ix, x, xii, 97, 98, 99, 105, 106, 107, 109,
starch, 199
110, 111, 116, 117, 118, 121, 124, 126, 128, 130,
statin, 148
131, 132, 133, 134, 135, 136, 137, 249, 251, 252,
statistics, 92
253, 254, 256, 258, 259, 261, 273, 275, 278, 280,
sterile, 216
281, 284, 285, 286, 287, 288
sterols, 142
target organs, 23, 63
stimulus, 215, 222
targets, x, 62, 181, 186, 188, 190, 191, 199, 202,
stock, 66, 316
265, 267, 268, 270
stomach, 5, 6, 8, 14, 15, 18, 24, 34, 40, 325, 328, 329
taxonomy, xiii, 291, 292, 293
stomatitis, 135, 136, 138
T-cell receptor, 244
storage, xiii, 19, 118, 214, 283, 301
technology, 292, 294
346 Index
temperature, viii, 23, 53, 58, 107, 215, 283 transport processes, 61, 92
tension, 252 trauma, 63
terpenes, 161 trial, 18, 29, 31, 46, 164, 165, 214, 225, 228
tetrahydrofurane, 133 triggers, 186, 211, 243, 256
Thailand, 296 triglycerides, 145, 233
therapeutics, 170, 188, 193 trypsin, 309
therapy, 29, 188, 189, 206, 244, 261, 270 tumor, xi, xii, 90, 91, 152, 153, 154, 155, 163, 175,
thermochemical cycle, 94 177, 178, 183, 184, 185, 186, 187, 188, 190, 191,
threonine, 244 193, 204, 206, 207, 208, 209, 210, 211, 213, 215,
threshold, 253, 304 235, 263, 270
thresholds, 189 tumor cells, 91, 154, 178, 183, 187, 193, 204, 206,
thrombin, 49 210, 211, 270
thymus, 163 tumor growth, 153, 175, 188, 206, 207, 208
thyroid, 150, 173 tumor invasion, 187
time, viii, xiv, 3, 6, 15, 18, 23, 28, 41, 62, 68, 97, 99, tumor metastasis, 187
107, 127, 128, 130, 132, 134, 146, 157, 166, 168, tumor necrosis factor, xi, 90, 155, 175, 186, 190,
175, 215, 217, 220, 241, 250, 278, 283, 292, 294, 209, 210, 213, 215
305, 306, 309, 312, 315, 323 tumor progression, 185, 187, 188
tissue, 6, 8, 11, 12, 23, 24, 36, 37, 38, 46, 145, 146, tumorigenesis, 179
174, 187, 195, 198, 222, 235, 237, 245 tumors, 152, 234
TNF, xi, 30, 155, 159, 191, 213, 215, 219, 222, 223, tumours, x, 141
266, 271 Turkey, 43
TNF-alpha, 159, 271 type 2 diabetes, xii, 237, 273, 274, 286, 287
tobacco, 156, 172 tyrosine, 86, 167, 179, 187, 235, 243, 244, 246, 265
tocopherols, 154 Tyrosine, 244
toluene, 25
tonic, xii, 273, 274, 285
topology, 55 U
total cholesterol, 143, 144
UK, 92, 93, 216, 297, 298, 301
toxic effect, 63, 158, 254
ulcer, 150, 177
toxicity, xii, 26, 55, 86, 89, 91, 93, 146, 152, 188,
ultrasound, 279
189, 193, 209, 254, 263
uncertainty, xiii, 291
toxicology, 62, 84, 86
uniform, 237
toxin, 155, 172, 254, 256, 260
United States, xiv, 327
trace elements, 234
updating, xi, 232
transcription, 48, 50, 63, 92, 184, 195, 196, 207, 210,
urea, 282
225, 227, 235, 236, 237, 238, 239, 244, 245, 246,
uric acid, 146
264
urinary bladder, 155
transcription factors, 196, 235, 236, 237, 245, 264
urinary bladder cancer, 155
transcripts, 195
urine, 2, 6, 8, 9, 12, 14, 15, 17, 18, 19, 20, 21, 38, 40,
transducer, 238, 253
42, 43, 45, 46, 47, 64, 187, 312
transduction, 203, 222, 239, 268
UV, 27, 59, 63, 127, 128, 163, 194, 199, 293, 296,
transfection, 195
298, 305, 316, 317, 318
transformation, 18, 29, 44, 51, 75, 195, 196, 321
UV irradiation, 127, 128
transformations, 74, 93, 324
UV light, 63, 199, 316, 317
transition, 121, 163, 171, 186, 234
UV spectrum, 318
transition metal, 163, 171, 234
uveitis, 158, 178
translation, 196
translocation, 235, 266, 323, 325
transmission, 177 V
transport, 5, 13, 22, 24, 35, 41, 54, 61, 62, 64, 82, 84,
92, 167, 168, 169, 170, 173, 176, 179, 237, 323, vacuum, 127, 128, 129, 130, 131, 132, 133, 317, 318
324, 326, 327, 328 vagina, 233
Index 347
Valencia, 201
validity, 31, 62, 296, 304
W
values, 7, 28, 62, 67, 75, 78, 80, 81, 82, 83, 84, 109,
weight gain, 29
118, 121, 124, 152, 160, 168, 169, 188, 201, 316,
wells, 216, 316
320, 324
West Africa, 321
variability, 39, 109, 218, 292
Western countries, 232, 233
variables, 23, 225
white blood cells, 160
variation, xiii, 30, 121, 233, 301, 302
wild type, 150
vas deferens, 149, 174
withdrawal, 258
vascular diseases, x, 181, 182
women, 9, 29, 32, 38, 46, 51, 207, 233
vascular endothelial growth factor (VEGF), 187
wood, 117, 118, 126, 195
vasodilation, 226
workers, 66
vasodilator, 223, 224
World Bank, 313
VCAM, 143, 176, 197
World Health Organisation, 303
vector, 239
wound healing, 63, 206
vegetables, vii, viii, xiv, 53, 54, 58, 89, 201, 234,
writing, 3
263, 266, 286, 287, 327
VEGF expression, 154, 176, 187, 206
VEGF protein, 154 X
vehicles, 199
velocity, 324 xenografts, 206
vertebrates, 47
vessels, 149
viruses, x, 98, 135, 136, 137, 138 Y
viscosity, 105, 107, 121
vitamin C, 89, 152, 177 yeast, 163, 164, 283
vitamin E, 89, 93, 94, 143, 161, 162, 173, 178 yield, 8, 37, 75, 100, 103, 109, 278, 317, 325
vitamins, 214
VLDL, 143